Homologous recombination-mediated transgene alterations in plants

ABSTRACT

The invention provides a process to prepare a recombined transgenic  Zea mays  plant from a transgenic  Zea mays  plant, wherein the transgene in the recombinant plant has an altered genetic structure relative to the genetic structure of the transgene in the transgenic plant, due to homologous recombination-mediated transgene deletion, amplification or rearrangement.

[0001] This application is a continuation-in-part of application Ser.No. 09/521,557 filed Mar. 9, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for producing transgeniccells, preferably plant cells, and transgenic plants in which thetransgene insertion has been altered by homologous recombination.Alterations include deletions, modifications, or duplications oftransgene sequences. The invention further relates to a method fordeleting ancillary sequences, such as selectable marker or reportergenes, from transgenic cells, preferably plant cells, and transgenicplants.

[0004] 2. Description of the Related Art

[0005] Genetically modified (GM) crops offer many advantages to thefarmer in terms of inputs to crop production, e.g. weed and insectcontrol, and improved usage of water and nutrient inputs. GM plants alsoprovide a means for improving nutritional value, e.g. improved aminoacid or protein composition, improved starch and oil quantities andqualities, increased vitamin levels, or bioavailability of nutrients, orcan be the source of pharmaceuticals or “nutraceuticals.” Methods havebeen developed for conferring tolerance or resistance to water or saltstress in monocots (U.S. Pat. No. 5,780,709), for example, and a singlegene has been used to improve tolerance to drought, salt loading, andfreezing in some plants (Kasuga et al., 1999). Insect resistance can beconferred by introducing genes for the production of toxins found in thesoil bacterium Bacillus thuringiensis (Bt). Lysine content has beenincreased by incorporating the genes for bacterial enzymes (e.g.Corynebacterium dihydropicolinic acid synthase and E. coliaspartokinase) into GM plants. The comparable plant enzymes are subjectto lysine feedback inhibition, while the bacterial enzymes show littleor no feedback inhibition.

[0006] Until technology made genetic modification of plants possible,production of plants with desirable characteristics was dependent uponselective breeding and the variability naturally present in a crop andclosely related sexually compatible species. Genetic modificationthrough transformation provides an efficient and controlled method forproducing plants with one or more desired characteristics, includingcharacteristics that are normally not found in those crops, such asresistance to herbicides or pests, or nutritionally balanced food orfeed products.

[0007] Genetic modification of crops by transformation sometimesinvolves transfer of one or more desired genes, along with ancillarysequences such as antibiotic resistance markers or reporter genes, intoa plant cell. Antibiotic resistance markers used in plant geneticengineering, for example, include the kanamycin resistance marker(Carrer et al., 1993), streptomycin resistance marker (Moll et al.,1990), lincomycin resistance marker (Jenkins et al., 1991) and theneomycin resistance marker (Beck et al., 1982). The ancillary sequencesare necessary for identification or selection of transformed cells, butdo not contribute to the trait conferred on the plant. Since ancillarysequences do not contribute to the desired crop improvement, effortshave been made to delete them from the GM progeny. Antibiotic resistancemarkers have particularly been targeted for deletion.

[0008] Furthermore, it has been demonstrated that using direct DNAdelivery methods, such as microprojectile bombardment, complex transgeneinsertions may occur including duplications, deletions, and complexrearrangements of introduced DNA (PCT Publication No. WO 99/32642).Complex transgene insertions may contribute to co-suppression of geneexpression or genetic instability of the insertion. Use of the presentinvention contributes to reducing the complexity of transgeneinsertions, thereby stabilizing gene expression and preferably removingancillary sequences.

[0009] A number of site-specific recombination-mediated methods havebeen developed for incorporating target genes into plant genomes, aswell as for deleting unwanted genetic elements from plant and animalcells. For example, the cre-lox recombination system of bacteriophageP1, described by Abremski et al. (1983), Stemberg et al. (1981) andothers, has been used to promote recombination in a variety of celltypes. The cre-lox system utilizes the cre recombinase isolated frombacteriophage P 1 in conjunction with the DNA sequences (termed loxsites) it recognizes. This recombination system has been effective forachieving recombination in plant cells (U.S. Pat. No. 5,658,772), animalcells (U.S. Pat. No. 4,959,317 and U.S. Pat. No. 5,801,030), and inviral vectors (Hardy et al., 1997).

[0010] Wahl et al. (U.S. Pat. No. 5,654,182) used the site-specific FLPrecombinase system of Saccharomyces cerevisiae to delete DNA sequencesin eukaryotic cells. The deletions were designed to accomplish eitherinactivation of a gene or activation of a gene by bringing desired DNAfragments into association with one another. Activity of the FLPrecombinase in plants has been demonstrated (Lyznik et al, 1996; Luo etal., 2000).

[0011] Others have used transposons, or mobile genetic elements thattranspose when a transposase gene is present in the same genome, toseparate target genes from ancillary sequences. Yoder et al. (U.S. Pat.No. 5,482,852 and U.S. Pat. No. 5,792,924) used constructs containingthe sequence of the transposase enzyme and the transposase recognitionsequences to provide a method for genetically altering plants thatcontain a desired gene free of vector and/or marker sequences.

[0012] Oliver et al. (U.S. Pat. No. 5,723,765) used site-specificrecombination systems in conjunction with a blocking sequence to providea regulatory mechanism in transgenic plants. In this method, whensite-specific recombination results in excision of the blockingsequence, regulatory elements that either induce or repress a particulargene of interest are moved into an appropriate position upstream fromthe target sequence.

[0013] Although each of these methods has been designed specifically toexcise unwanted sequences, each also relies upon introduction ofancillary genetic sequences (e.g., recombinase or transposase specificrecognition sequences) that ultimately do not contribute to the desiredcrop improvement.

[0014] Thus, there is a need for a method for excising unwanted DNAsequences from transgenic cells without introducing any furtherancillary DNA sequences.

[0015] The present invention is exemplified herein by alterations oftransgenic insertions in plant cells and transgenic plants. It is,however, the belief of the inventors that the methods of the presentinvention are equally applicable to, and useful in, any organism inwhich homologous recombinatin of DNA occurs.

SUMMARY OF THE INVENTION

[0016] The invention provides a novel method for excision, modification,or amplification of DNA sequences from transgenic cells that does notinvolve the use of site-specific recombination enzymes, includingtransposase enzymes, but instead relies upon directly repeated DNAsequences positioned about the target sequence to direct excision oramplification through native cellular recombination mechanisms. Theinvention provides a method of preparing a recombined transgenic cellhaving a preselected DNA sequence flanked by directly repeated DNAsequences. Additionally, the transgene insertion may comprise furtherDNA sequences. In the method of the present invention, the direct repeatmay be recognized by a site-specific recombinase enzyme, but asite-specific recombinase is not required for deletion of the desiredsequence.

[0017] The invention provides a method of preparing a transgenic cellhaving an altered transgene insertion. A first transgenic cell isobtained, wherein the transgenic insertion DNA sequence comprises apre-selected DNA sequence flanked by directly repeated DNA sequences. Aplurality of progeny cells of any generation are obtained and a secondcell is identified from a the progeny cells, wherein the second cellcontains a DNA insertion sequence that has been altered byrecombination. The first cell can be either homozygous or hemizygous forthe second DNA sequence.

[0018] The invention further provides methods of using a negativeselectable marker gene to identify cells with altered transgeneinsertions.

[0019] The invention provides a novel method of removing undesirable DNAsequences as well as a method for resolving complex transgene insertionsto simpler insertions, thereby increasing transgene stability anddecreasing the occurrence of co-suppression.

[0020] The invention provides a method of preparing a fertile transgenicplant having an altered transgene insertion comprising obtaining a firstfertile transgenic plant comprising a transgene insertion DNA sequence,wherein the transgene insertion DNA sequence comprises a pre-selectedDNA sequence flanked by directly repeated DNA sequences, obtaining aplurality of progeny of any generation of the first transgenic plant,and selecting a progeny fertile transgenic plant wherein the transgeneinsertion is altered as compared to the first fertile transgenic plant.Methods are provided wherein the pre-selected DNA sequence comprises aselectable marker gene or a reporter gene, such as a bar, nptII or agene encoding a glyphosate resistant EPSPS enzyme. Furthermore, methodsare provided wherein the plurality of progeny plants are obtained byeither self-pollination or outcrossing. The resultant progeny plants maybe either inbreds or hybrids. The plants may be monocot plants, such asa maize, sorghum, barley, wheat rye or rice or dicot plants such assoybean, canola, sunflower, or cotton.

[0021] The invention provides a method of preparing a recombined fertiletransgenic plant, by obtaining a first fertile transgenic plant having apreselected DNA sequence flanked by directly repeated DNA sequences.Additionally, the transgene insertion may comprise further DNAsequences. In the method of the present invention, the direct repeat maybe recognized by a site-specific recombinase enzyme, but a site-specificrecombinase is not required for deletion of the desired sequence. Thefirst fertile transgenic plants are crossed to produce either hybrid orinbred progeny plants, and from those progeny plants one or more secondfertile transgenic plants are selected that contain a second DNAsequence that has been altered by recombination. The first fertiletransgenic plant can be either homozygous or hemizygous for the secondDNA sequence.

[0022] Also provided by the present invention is a transgenic cell orplant produced by the method, wherein the transgene insertion is alteredas compared to the first fertile transgenic cell or plant.

[0023] The invention also provides a seed for producing a recombinanttransgenic plant, wherein the transgene insertion is altered as comparedto a first fertile transgenic plant.

[0024] Also provided is a fertile transgenic plant wherein a transgeneinsertion is altered from a parent transgene insertion. The plant may behybrid or inbred. The transgene insertion may be altered in that it hasbeen deleted, amplified, or rearranged.

[0025] Further provided is a progeny cell or plant of any generationcomprising an altered transgene insertion, wherein the transgeneinsertion is altered from the transgene insertion in a parental R₀plant.

[0026] The present invention also provides an altered transgeneinsertion DNA sequence preparable by the method comprising obtaining afirst fertile transgenic plant comprising a transgene insertion DNAsequence, wherein the transgene DNA sequence comprises a pre-selectedDNA sequence flanked by directly repeated DNA sequences; obtaining aplurality of progeny of any generation of the first transgenic plant;and selecting a progeny fertile transgenic plant wherein the transgeneinsertion is altered as compared to the first fertile transgenic plant.The transgene insertion may be altered in that it has been deleted,amplified, or rearranged. Alteration of the transgene insertion mayresult in a change in expression of a transgene contained within theparental transgene insertion. The alteration of the transgene may beidentified by DNA analysis, such as by PCR or Southern blot analysis.The altered transgene insertion may be in a monocot plant, such as amaize, sorghum, barley, wheat, rye or rice plant or a dicot plant suchas cotton, soybean, sunflower or canola.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1. Pathways for obtaining deletion derivatives

[0028]FIG. 2. Gene conversion pathway (non-reciprocal recombination) forobtaining deletion derivatives.

[0029]FIG. 3. Single strand annealing model.

[0030]FIG. 4. Plasmid vector pMON19344

[0031]FIG. 5. Direct repeat induced homologous recombination-mediatedalteration of a transgene insertion.

[0032]FIG. 6. Plasmid vector pDPG354

[0033]FIG. 7. Plasmid vector pDPG165

[0034]FIG. 8. Plasmid vector pDPG320

[0035]FIG. 9. DBT418 transgene insertion indicating direct repeatsequences that were the substrate for homologous recombination productsrecovered in the 09-07 and 03-09 altered transgene insertion progeny.

[0036]FIG. 10. Altered transgene insertions recovered followinghomologous recombination in DBT418.

[0037]FIG. 11. MON849 transgene insertion and altered insertionsrecovered following homologous recombination.

[0038]FIG. 12. Plasmid vector pMON36133.

[0039]FIG. 13. Use of a negative selectable marker to select for analtered transgene insertion.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The invention provides a novel method for production oftransgenic cells and plants lacking ancillary DNA sequences that do notcontribute to the desired phenotypic trait. The inventors havediscovered that homologous recombination occurs in the plant cells at arate sufficient to provide recombined transgenic progeny without addedrecombinase enzymes. In the method provided, directly repeated DNAsequences are located 5′ and 3′ to a target sequence, to be amplifiedwithin, modified, or excised from the plant genome. The inventors havedetermined that gene deletion frequency, for example, is approximately0.1% per 287±19 base pairs of homologous direct repeat sequence. Themethod described herein can be used to delete selectable marker genes,to delete partial or rearranged gene copies, to reduce overall transgenecopy number, or to increase overall transgene copy number.

[0041] In direct repeat-mediated homologous recombination, which can beused particularly to produce transgene deletions, direct repeats thatare present in the introduced DNA sequence, or produce a DNA alignment,result in amplification of the number of copies of a particular genesequence or excision of either one or more ancillary DNA sequences ortarget DNA sequences. This method can be used to delete ancillarysequences or to remove regulatory sequences, for example, in order toactivate or deactivate a target gene.

[0042] It is known that homologous recombination results in geneticrearrangements of transgenes in plants. Repeated DNA sequences have beenshown to lead to deletion of a flanked sequence in various dicotspecies, e.g. Arabidopsis thaliana (Swoboda et al., 1994); Jelesko etal., 1999), Brassica napus (Gal et al., 1991; Swoboda et al, 1993) andNicotiana tabacum (Peterhans et al., 1990; Zubko et al., 2000). In mostinstances alteration of transgene sequences was attributed torecombination in somatic cells and occurred at low frequencies generallyranging from 10⁻⁶ to 10⁻⁷. Meiotic recombination of a native plantsequence transgene was observed in Arabidopsis at a frequency of 3×10⁻⁶and in tobacco at a frequency of 3×10⁻⁵ to 1×10⁻⁶ (Tovar andLichtenstein, 1992). In tobacco the more frequent recombinants wererecovered from homozygous parents (Tovar and Lichtenstein, 1992), butonly at a frequency of about 3×10⁻⁵. While it is possible to screenmillions of seeds to identify alterations of transgene sequences inspecies such as Arabidopsis thaliana or Nicotiana tabacum, in agronomiccrops such as corn screening of millions of plants for loss ofexpression of a transgene would be difficult. For example, a fieldscreen of 1,000,000 corn plants would require about 40 acres of plantsto be screened to identify a single recombinant at a frequency of 10⁻⁶.In the method of the present invention, homologous recombination oftransgenes was observed at frequencies greater than 0.1% and, therefore,a screen of recombinants could be conducted on as few as 1000 plantsrequiring only about 0.04 acres in the field.

[0043] One of the most widely held models for homologous recombinationis the double-strand break repair (DSBR) model (Szostak et al, 1983). Inthe context of the DSBR model, there are three reciprocal recombinationpathways for generating a deletion by recombination between directrepeats. These are shown in FIG. 1. Directly repeated DNA is representedby the small dotted rectangles in FIG. 1. In the first pathway (FIG.1A), commonly referred to as a “loop out”, the chromatid loops back onitself and a reciprocal exchange in the region of the homology resultsin excision of a circle bearing one of the two repeats. Loop outs havebeen widely observed in a variety of systems, and can occur betweenrepeats that are very closely linked, i.e. less than 1 Kb apart. Thus,there is no steric hindrance to loop outs between most transgenerepeats. The next two pathways, unequal sister chromatid crossover (FIG.1B) and unequal inter-homologue crossover (FIG. 1C) are identical exceptthat in the former recombination occurs between sister chromatids and inthe latter it occurs between chromosomes pairs. In both cases thereciprocality results in a deletion on one chromatid and an increase incopy number on the other. In the case of unequal inter-homologuecrossovers (FIG. 1C), flanking alleles will be recombined. All pathwaysare examples of reciprocal recombination. Even in the example of theloop outs, it is clear that this process is reciprocal, although one ofthe two products (the excised circle) will be lost in subsequent celldivisions.

[0044] The DSBR model can give rise to reciprocal recombination eventssuch as those shown above, as well as nonreciprocal recombination eventsknown as gene conversions. Gene conversion can occur frequently betweentransgene repeats. Evidence for gene conversion between inverted repeatsin plants was obtained by Tovar and Lichtenstein (1992). Deletion byreciprocal recombination was not possible in this system (since therepeats were inverted, not direct), but it may be possible to obtain adeletion by a gene conversion pathway. An example of this is shown inFIG. 2. For convenience the example uses the context of the DSBR model,although other models may apply. If a double strand break occurs in orbetween two repeated elements on one chromatid (FIG. 2A), the DSB can beexpanded into a gap reaching the sequences of the two direct repeats(FIG. 2B), deleting the intervening sequence. The gap can then berepaired using one of the two repeats on another chromatid as atemplate. The repair product will have deleted one of the repeats on thechromatid on which the DSB initiated (FIG. 2C), without a concomitantincrease in copy number on the other chromatid, i.e. the event wasnonreciprocal.

[0045] Both types of recombination described above, reciprocal andnonreciprocal, are conservative recombination pathways, i.e. there is nophysical loss of DNA sequence in the final products relative to theparental molecules. Genetic information may have changed, but thechromosomes still have the same general physical structure. Evidence fora nonconservative pathway exists. Experiments in yeast have led to theproposal of a model to account for nonconservative recombination betweenclosely linked direct repeats. This model, called the single-strandannealing (SSA) model, is shown in FIG. 3.

[0046] In the SSA model, recombination between closely linked directrepeats initiates with a DSB between the two regions of homology (asshown in FIG. 3) or within one of the two repeats. As in the DSBR model,the ends are processed by an exonuclease to generate longsingle-stranded tails. As the tails extend into the regions of homologycomplementary DNA is revealed, allowing the two tails to anneal to eachother. Any sequences that were between the two repeats would be left assingle stranded tails and would be removed, perhaps by a secondnuclease. After ligation of nicks the final product has deleted the DNAbetween the two repeats in a nonconservative manner, i.e. theintervening DNA is lost in the process. As with the DSBR repair model,significant experimental evidence, primarily from yeast, exists tosupport the SSA model.

[0047] There are important distinctions between the DSBR model and theSSA model. First, the SSA model will work only with direct repeats ofhomology, whereas the DSBR model will work with either inverted ordirect repeats, although only in the latter will a deletion occur.Second, the DSBR mechanism can occur within a chromatid (i.e. shownbelow in FIG. 1A) or between two chromatids (i.e. shown below in FIGS.1B and 1C). With the SSA mechanism, recombination is likely to involveonly one chromatid. In order for recombination to take place between twochromatids both chromatids would have to sustain a DSB in approximatelythe same position at approximately the same time

[0048] In summary, there are at least five pathways by which deletionscan be formed by homologous recombination between direct repeats. Threepathways involve reciprocal recombination (FIG. 1), one pathway involvesnonreciprocal recombination (gene conversion, FIG. 2), and one pathwayis nonconservative, the SSA model (FIG. 3). Table 1 summarizes thesepathways and their characteristics. TABLE 1 Pathways for obtainingdeletion derivatives in plants other distinguishing Pathway effect ofzygosity properties loop out deletion can occur in extrachromosomalcircle is hemi- or homozygotes produced, no increase in copy numberunequal sister deletion can occur in increase in copy number onchromatid hemi- or homozygotes sister chromatid exchange unequal inter-homozygosity is required increase in copy number on homologue fordeletions to occur homologue, flanking exchange markers recombined, maybe elevated in meiosis gene conversion deletion can occur in no increasein copy hemi- or homozygotes number, flanking markers are notrecombined, may be elevated in meiosis in homozygotes single stranddeletion can occur in may be distance dependent, annealing hemi- orhomozygotes i.e. closer repeats recombine more

[0049] By producing a transgene construct that incorporates DNA sequencehomologies at desired locations, it is possible to enhance the frequencyof such homologous recombination events in transgenic plant cells,resulting in targeted deletion or amplification of desired DNA sequencesin progeny cells.

[0050] The method of the present invention can be used with a variety ofplants, and is especially useful for development of transgenic monocotplants, such as maize, sorghum, barley, wheat, rye or rice and dicotplants such as soybean, cotton, canola and potato.

[0051] II. Definitions

[0052] The following words and phrases have the meanings set forthbelow.

[0053] Chimeric gene: A gene or DNA sequence or segment comprising atleast two DNA sequences or segments from species that do not combine DNAunder natural conditions, or DNA sequences or segments that arepositioned or linked in a manner that does not normally occur in thenative genome of an untransformed plant, such as maize or anothermonocot.

[0054] Exogenous gene: A gene that is not normally present in a givenhost genome in the present form. In this respect, the gene itself may benative to the host genome, however the exogenous gene will comprise thenative gene altered by the addition or deletion of one or more differentregulatory elements.

[0055] Expression: An intracellular process undergone by a coding DNAmolecule, such as a structural gene, to produce at least an RNAmolecule. Usually a polypeptide is produced through the combinedprocesses of transcription and translation.

[0056] Expression cassette: A nucleic acid segment comprising at least afirst gene that one desires to have expressed in a host cell and thenecessary regulatory elements for expressing the gene in the host cell.Preferred regulatory elements for use with the invention includepromoters, enhancers and terminators. It also may be desirable toinclude on the expression cassette a nucleic acid segment encoding anappropriate transit peptide, as is described below. The expressioncassette may be contained and propagated in any suitable cloning vector,for example, a plasmid, cosmid, bacterial artificial chromosome, oryeast artificial chromosome. The whole vector DNA may be used totransform a host cell, or alternatively, the expression cassette may beisolated from the vector and then used for transformation.

[0057] Expression vector: A vector comprising at least one expressioncassette.

[0058] Heterologous DNA: DNA from a source different from that of therecipient cell.

[0059] Homologous DNA: DNA from the same source as that of the recipientcell.

[0060] Obtaining: When used in conjunction with a transgenic plant cellor transgenic plant, obtaining means either transforming anon-transgenic plant cell or plant to create the transgenic plant cellor plant, or planting transgenic plant seed to produce the transgenicplant cell or plant.

[0061] Progeny: Any subsequent generation, including the seeds andplants therefrom, which is derived from a particular parental plant orset of parental plants.

[0062] Promoter: A recognition site on a DNA sequence or group of DNAsequences that provide an expression control element for a structuralgene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene.

[0063] R₀ Transgenic Plant: A plant that has been directly transformedwith selected DNA or has been regenerated from a cell or cell clusterthat has been transformed with a selected DNA.

[0064] Recombined transgenic: A transgenic plant cell, plant part, planttissue or plant, the transgenic DNA sequences or genes of which arealtered by non-reciprocal homologous recombination. Altered includesdeleted, amplified, or any other modification of the preselected DNAsequence as originally integrated into the host genome.

[0065] Regeneration: The process of growing a plant from a plant cell(e.g., plant protoplast, callus or explant).

[0066] Selected DNA: A DNA segment that one desires to introduce into aplant genome by genetic transformation.

[0067] Selected gene: A gene that one desires to have expressed in atransformed plant, plant cell or plant part. A selected gene may benative or foreign to a host genome, but where the selected gene isnative to the host genome, will include one or more regulatory orfunctional elements that differ from native copies of the gene.

[0068] Structural gene: A gene that is expressed to produce apolypeptide.

[0069] Transformation: A process of introducing an exogenous DNAsequence (e.g., a vector, a recombinant DNA molecule) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

[0070] Transformation construct: A chimeric DNA molecule that isdesigned for introduction into a host genome by genetic transformation.Preferred transformation constructs will comprise all of the geneticelements necessary to direct the expression of one or more exogenousgenes. In particular embodiments of the instant invention, it may bedesirable to introduce a transformation construct into a host cell inthe form of an expression cassette.

[0071] Transformed cell: A cell wherein its DNA has been altered by theintroduction of an exogenous DNA molecule into that cell.

[0072] Transgene: A segment of DNA that has been incorporated into ahost genome or is capable of autonomous replication in a host cell andis capable of causing the expression of one or more cellular products.Exemplary transgenes will provide the host cell, or plants regeneratedtherefrom, with a novel phenotype relative to the correspondingnon-transformed cell or plant. Transgenes may be directly introducedinto a plant by genetic transformation, or may be inherited from a plantof any previous generation that was transformed with the DNA segment.

[0073] Transgene insertion: A segment of DNA incorporated into a hostgenome. A transgene insertion comprises all of the DNA sequences thatwere introduced by transformation and are present at a single geneticlocus in a transformed cell or plant. DNA sequences within the transgeneinsertion may arise from one or more plasmid vectors. Furthermore, DNAsequences may be rearranged in a transgene insertion when compared tothe arrangement of DNA sequences in the parent plasmid vector orvectors. A transgene insertion may be altered using the methods of thisinvention, resulting in deletion, duplication, or rearrangement of DNAsequences. A parent transgene insertion is the original transgeneinsertion in a parent plant. The parent transgene insertion may bealtered by non-reciprocal recombination during a cycle of meiosis andthen transmitted to the progeny as an altered transgene insertion.

[0074] Transgenic cell: Any cell derived or regenerated from atransformed cell or derived from a transgenic cell. Exemplary transgeniccells include plant calli derived from a transformed plant cell andparticular somatic cells such as leaf, root, stem, or reproductive(germ) cells obtained from a transgenic plant.

[0075] Transit peptide: A polypeptide sequence that is capable ofdirecting a polypeptide to a particular organelle or other locationwithin a cell.

[0076] Vector: A DNA molecule capable of replication in a host celland/or to which another DNA segment can be operatively linked so as tobring about replication of the attached segment. A plasmid is anexemplary vector.

[0077] Wild type: An untransformed plant cell, plant part, plant tissueor plant wherein the genome has not been altered by the presence of apreselected DNA sequence.

[0078] II. DNA Constructs of the Invention

[0079] Virtually any DNA may be used for delivery to recipient cells toultimately produce fertile transgenic plants in accordance with thepresent invention. For example, an isolated and purified DNA segment inthe form of vectors and plasmids encoding a desired gene product orlinear DNA fragments, in some instances containing only the DNA elementto be expressed in the plant, and the like, may be employed.

[0080] DNA useful for introduction into plant cells includes that whichhas been derived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into a plant. An example of DNA “derived” from asource, would be a DNA sequence or segment that is identified as auseful fragment within a given organism, and is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.Such DNA is commonly referred to as “recombinant DNA.”

[0081] Therefore, useful DNA includes completely synthetic DNA,semi-synthetic DNA, DNA isolated from biological sources, and DNAderived from RNA. It is within the scope of the invention to isolate andpurify a DNA segment from a given genotype, and to subsequentlyintroduce multiple copies of the isolated and purified DNA segment intothe same genotype, e.g., to enhance production of a given gene product.

[0082] The introduced DNA includes, but is not limited to, DNA fromplant genes and non-plant genes, such as those from bacteria, yeasts,animals or viruses. The introduced DNA can include modified genes,portions of genes, or chimeric genes, including genes from the same ordifferent genotype.

[0083] An isolated and purified DNA segment, molecule or sequence can beidentified and isolated by standard methods, as described by Sambrook etal. (1989). The isolated and purified DNA segment can be identified byscreening of a DNA or cDNA library generated from nucleic acid derivedfrom a particular cell type, cell line, primary cells, or tissue.Screening for DNA fragments that encode all or a portion of the isolatedand purified DNA segment can be accomplished by screening plaques from agenomic or cDNA library for hybridization to a probe of the DNA fromother organisms or by screening plaques from a cDNA expression libraryfor binding to antibodies that specifically recognize the proteinencoded by the isolated and purified DNA segment. DNA fragments thathybridize to an isolated and purified DNA segment probe from otherorganisms and/or plaques carrying DNA fragments that are immunoreactivewith antibodies to the protein encoded by the isolated and purified DNAsegment can be subcloned into a vector and sequenced and/or used asprobes to identify other cDNA or genomic sequences encoding all or aportion of the isolated and purified DNA segment.

[0084] Portions of the genomic copy or copies of the isolated andpurified DNA segment can be sequenced and the 5′ end of the DNAidentified by standard methods including either DNA sequence homology toother homologous genes or by RNAase protection analysis, as described bySambrook et al. (1989). Once portions of the 5′ end of the isolated andpurified DNA segment are identified, complete copies of the isolated andpurified DNA segment can be obtained by standard methods, includingcloning or polymerase chain reaction (PCR) synthesis usingoligonucleotide primers complementary to the isolated and purified DNAsegment at the 5′ end of the DNA. The presence of an isolatedfull-length copy of the isolated and purified DNA can be verified byhybridization, partial sequence analysis, or by expression of theisolated and purified DNA segment.

[0085] The DNA may be circular or linear, double-stranded orsingle-stranded. Generally, the DNA is in the form of chimeric DNA thatcan also contain coding regions flanked by regulatory sequences thatpromote the expression of the recombinant DNA present in the resultantplant (an “expression cassette”). For example, the DNA may itselfcomprise or consist of a promoter that is active in which is derivedfrom a non-source, or may utilize a promoter already present in thegenotype.

[0086] Ultimately, the most desirable DNA segments for introduction intoa monocot genome may be homologous genes or gene families that encode adesired trait (e.g., increased yield per acre) and that are introducedunder the control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue-specific (e.g., root-, collar/sheath-, whorl-,stalk-, ear shank-, kernel- or leaf-specific) promoters or controlelements. Indeed, it is envisioned that a particular use of the presentinvention may be the targeting of an isolated and purified DNA segmentin a tissue- or organelle-specific manner.

[0087] The construction of vectors that may be employed in conjunctionwith the present invention will be known to those of skill in the art inlight of the present disclosure (see, e.g., Sambrook et al., 1989;Gelvin et al., 1990).

[0088] Generally, the introduced DNA will be relatively small, i.e.,less than about 30 kb to minimize any susceptibility to physical,chemical, or enzymatic degradation that is known to increase as the sizeof the DNA increases. The number of proteins, RNA transcripts ormixtures thereof that is introduced into the genome is preferablyisolated and purified and defined, e.g., from one to about 5-10 suchproducts of the introduced DNA may be formed.

[0089] A. Expression Cassettes

[0090] 1. Promoters, Enhancers and Other Non-3¢ Transcription RegulatorySequences

[0091] Preferably, the expression cassette of the invention is operablylinked to a promoter, which provides for expression of a linked DNAsequence. The DNA sequence is operably linked to the promoter when it islocated downstream from the promoter, to form an expression cassette. Anisolated promoter sequence that is a strong promoter for heterologousDNAs is advantageous because it provides for a sufficient level of geneexpression to allow for easy detection and selection of transformedcells and provides for a high level of gene expression when desired.

[0092] Most endogenous genes have regions of DNA that are known aspromoters, which regulate gene expression. Promoter regions aretypically found in the flanking DNA upstream from the coding sequence inboth prokaryotic and eukaryotic cells. A promoter sequence provides forregulation of transcription of the downstream gene sequence andtypically includes from about 50 to about 2,000 nucleotide base pairs.Promoter sequences also contain regulatory sequences such as enhancersequences that can influence the level of gene expression. Some isolatedpromoter sequences can provide for gene expression of heterologous DNAs,that is a DNA different from the native or homologous DNA.

[0093] Promoter sequences are also known to be strong or weak orinducible. A strong promoter provides for a high level of geneexpression, whereas a weak promoter provides for a very low level ofgene expression. An inducible promoter is a promoter that provides forthe turning on and off of gene expression in response to an exogenouslyadded agent, or to an environmental or developmental stimulus. Promoterscan also provide for tissue specific or developmental regulation.

[0094] Preferred expression cassettes will generally include, but arenot limited to, a plant promoter such as the CaMV 35S promoter (Odell etal., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebertet al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang et al.,1990), α-tubulin, ubiquitin, actin (Wang et al., 1992), cab (Sullivan etal., 1989), PEPCase (Hudspeth et al., 1989) or those associated with theR gene complex (Chandler et al., 1989). Further suitable promotersinclude inducible promoters, such as the light inducible promoterderived from the pea rbcS gene (Coruzzi et al., 1971), the actinpromoter from rice (McElroy et al., 1990), and water-stress-, ABA-andturgor-inducible promoters (Skriver et al., 1990; Guerrero et al.,1990), tissue-specific promoters, such as root-cell promoters (Conklinget al., 1990), and developmentally-specific promoters such as seedspecific promoters, e.g., the phaseolin promoter from beans(Sengupta-Gopalan, 1985), and the Z10 and Z27 promoters from maize. Forexample, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination. Tissue specific expressionmay also be functionally accomplished by introducing a constitutivelyexpressed gene (all tissues) in combination with an antisense gene thatis expressed only in those tissues where the gene product is notdesired.

[0095] Promoters that direct specific or enhanced expression in certainplant tissues will be known to those of skill in the art in light of thepresent disclosure. These include, for example, the rbcS promoter,specific for green tissue; the ocs, nos and mas promoters that havehigher activity in roots or wounded leaf tissue; a truncated (−90 to +8)35S promoter that directs enhanced expression in roots, an α-tubulingene that directs expression in roots and promoters derived from zeinstorage protein genes that direct expression in endosperm. Transcriptionenhancers or duplications of enhancers can be used to increaseexpression from a particular promoter (see, for example, Fromm et al.,1989). Examples of such enhancers include, but are not limited to,elements from the CaMV 35S promoter and octopine synthase genes (U.S.Pat. No. 5,290,924). It is particularly contemplated that one mayadvantageously use the 16 bp ocs enhancer element from the octopinesynthase (ocs) gene (Ellis et al., 1987; Bouchez et al., 1989),especially when present in multiple copies, to achieve enhancedexpression in roots. Other promoters useful in the practice of theinvention are known to those of skill in the art. For example, see VanOoijen et al. (U.S. Pat. No. 5,593,963) and Walsh et al. (U.S. Pat. No.5,743,477).

[0096] Alternatively, novel tissue-specific promoter sequences may beemployed in the practice of the present invention. cDNA clones from aparticular tissue are isolated and those clones that are expressedspecifically in that tissue are identified, for example, using Northernblotting. Preferably, the gene isolated is not present in a high copynumber, but is expressed in specific tissues. The promoter and controlelements of corresponding genomic clones can then be localized usingtechniques well known to those of skill in the art.

[0097] As the DNA sequence inserted between the transcription initiationsite and the start of the coding sequence, i.e., the untranslated leadersequence, can influence gene expression, one can also employ aparticular leader sequence. Preferred leader sequence include those thatcomprise sequences selected to direct optimum expression of the attachedgene, i.e., to include a preferred consensus leader sequence that canincrease or maintain mRNA stability and prevent inappropriate initiationof translation (Joshi, 1987). Such sequences are known to those of skillin the art. Sequences that are derived from genes that are highlyexpressed in plants and in maize, in particular, will be most preferred.

[0098] Regulatory elements such as Adh intron 1 (Callis et al., 1987),sucrose synthase intron (Vasil et al., 1989), rice actin 1 intron 1(McElroy et al., 1991) or TMV omega element (Gallie et al., 1989) canalso be included where desired. Other such regulatory elements useful inthe practice of the invention are known to those of skill in the art.

[0099] An isolated and purified DNA segment can be combined with thetranscriptional regulatory sequences by standard methods as described inSambrook et al., cited supra, to yield an expression cassette. Briefly,a plasmid containing a promoter such as the 35S CaMV promoter can beconstructed as described in Jefferson (1987) or obtained from ClontechLab in Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, theseplasmids are constructed to provide for multiple cloning sites havingspecificity for different restriction enzymes downstream from thepromoter. The isolated and purified DNA segment can be subcloneddownstream from the promoter using restriction enzymes to ensure thatthe DNA is inserted in proper orientation with respect to the promoterso that the DNA can be expressed. Once the isolated and purified DNAsegment is operably linked to a promoter, the expression cassette soformed can be subcloned into a plasmid or other vectors.

[0100] 2. Targeting Sequences

[0101] Additionally, expression cassettes can be constructed andemployed to target the product of the isolated and purified DNA sequenceor segment to an intracellular compartment within plant cells or todirect a protein to the extracellular environment. This can generally beachieved by joining a DNA sequence encoding a transit or signal peptidesequence to the coding sequence of the isolated and purified DNAsequence. The resultant transit, or signal, peptide will transport theprotein to a particular intracellular, or extracellular destination,respectively, and can then be post-translationally removed. Transitpeptides act by facilitating the transport of proteins throughintracellular membranes, e.g., vacuole, vesicle, plastid andmitochondrial membranes, whereas signal peptides direct proteins throughthe extracellular membrane. By facilitating transport of the proteininto compartments inside or outside the cell, these sequences canincrease the accumulation of a particular gene product in a particularlocation. For example, see U.S. Pat. Nos. 5,258,300 and 5,593,963.

[0102] The isolated and purified DNA segment can be directed to aparticular organelle, such as the chloroplast rather than to thecytoplasm. Thus, the expression cassette can further be comprised of achloroplast transit peptide encoding DNA sequence operably linkedbetween a promoter and the isolated and purified DNA segment (for areview of plastid targeting peptides, see Heijne et al. (1989); Keegstraet al. (1989). This is exemplified by the use of the rbcS (RuBISCO)transit peptide that targets proteins specifically to plastids.

[0103] A chloroplast transit peptide can be used. A chloroplast transitpeptide is typically 40 to 70 amino acids in length and functionspost-translationally to direct a protein to the chloroplast. The transitpeptide is cleaved either during or just after import into thechloroplast to yield the mature protein. The complete copy of theisolated and purified DNA segment may contain a chloroplast transitpeptide sequence. In that case, it may not be necessary to combine anexogenously obtained chloroplast transit peptide sequence into theexpression cassette.

[0104] Chloroplast transit peptide encoding sequences can be obtainedfrom a variety of plant nuclear genes, so long as the products of thegenes are expressed as preproteins comprising an amino terminal transitpeptide and transported into chloroplast. Examples of plant geneproducts known to include such transit peptide sequences include, butare not limited to, the small subunit of ribulose biphosphatecarboxylase, ferredoxin, chlorophyll a/b binding protein, chloroplastribosomal proteins encoded by nuclear genes, certain heat shockproteins, amino acid biosynthetic enzymes such as acetolactate acidsynthase, 3-enolpyruvylphosphoshikimate synthase, dihydrodipicolinatesynthase, and the like. Alternatively, the DNA fragment coding for thetransit peptide may be chemically synthesized either wholly or in partfrom the known sequences of transit peptides such as those listed above.Furthermore, the transit peptide may compromise sequences derived fromtransit peptides from more than one source and may include a peptidesequence derived from the amino-terminal region of the mature proteinthat in its native state is linked to a transit peptide, e.g., see U.S.Pat. No. 5,510,471.

[0105] Regardless of the source of the DNA fragment coding for thetransit peptide, it should include a translation initiation codon and anamino acid sequence that is recognized by and will function properly tofacilitate import of a polypeptide into chloroplasts of the host plant.Attention should also be given to the amino acid sequence at thejunction between the transit peptide and the protein encoded by theisolated and purified DNA segment where it is cleaved to yield themature enzyme. Certain conserved amino acid sequences have beenidentified and may serve as a guideline. Precise fusion of the transitpeptide coding sequence with the isolated and purified DNA segmentcoding sequence may require manipulation of one or both DNA sequences tointroduce, for example, a convenient restriction site. This may beaccomplished by methods including site-directed mutagenesis, insertionof chemically synthesized oligonucleotide linkers, and the like.

[0106] Once obtained, the chloroplast transit peptide sequence can beappropriately linked to the promoter and the isolated and purified DNAsegment in an expression cassette using standard methods. Briefly, aplasmid containing a promoter functional in plant cells and havingmultiple cloning sites downstream can be constructed as described inJefferson, cited supra. The chloroplast transit peptide sequence can beinserted downstream from the promoter using restriction enzymes. Theisolated and purified DNA segment can then be inserted immediatelydownstream from and in frame with the 3¢ terminus of the chloroplasttransit peptide sequence so that the chloroplast transit peptide islinked to the amino terminus of the protein encoded by the isolated andpurified DNA segment. Once formed, the expression cassette can besubcloned into other plasmids or vectors.

[0107] Alternatively, targeting of the gene product to an intracellularcompartment within plant cells may also be achieved by direct deliveryof an isolated and purified DNA segment to the intracellularcompartment. For example, an expression cassette encoding a protein, thepresence of which is desired in the chloroplast, may be directlyintroduced into the chloroplast genome using the method described inU.S. Pat. No. 5,451,513.

[0108] It may be useful to target DNA itself within a cell. For example,it may be useful to target an introduced isolated and purified DNA tothe nucleus, as this may increase the frequency of transformation.Nuclear targeting sequences that function in plants are known, e.g., theAgrobacterium virD protein is known to target DNA sequences to thenucleus of a plant cell (Herrera-Estrella et al., 1990). Within thenucleus itself, it would be useful to target a gene in order to achievesite-specific integration. For example, it would be useful to have agene introduced through transformation replace an existing gene in thecell.

[0109] 3. 3′ Sequences

[0110] When the expression cassette is to be introduced into a plantcell, the expression cassette can also optionally include 3′nontranslated plant regulatory DNA sequences that act as a signal toterminate transcription and allow for the polyadenylation of theresultant mRNA. The 3′ nontranslated regulatory DNA sequence preferablyincludes from about 300 to 1,000 nucleotide base pairs and containsplant transcriptional and translational termination sequences.

[0111] Preferred 3′ elements are derived from those from the nopalinesynthase gene of Agrobacterium tumefaciens (Bevan et al., 1983), theterminator for the T7 transcript from the Agrobacterium tumefaciens,T-DNA and the 3′ end of the protease inhibitor I or II genes from potatoor tomato, although other 3′ elements known to those of skill in the artcan also be employed. These 3′ nontranslated regulatory sequences can beobtained as described in Methods in Enzymology (1987) or are alreadypresent in plasmids available from commercial sources such as Clontech(Palo Alto, Calif.). The 3′ nontranslated regulatory sequences can beoperably linked to the 3′ terminus of the isolated and purified DNAsegment by standard methods.

[0112] 4. Marker Genes

[0113] In order to improve the ability to identify transformants, onemay desire to employ one or more selectable marker genes or reportergenes as, or in addition to, the expressible isolated and purified DNAsegment(s). “Marker genes” or “reporter genes” are genes that impart adistinct phenotype to cells expressing the marker gene and thus allowsuch transformed cells to be distinguished from cells that do not havethe gene. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait that one can“select” for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a “reporter” trait that one can identify through observation ortesting, i.e., by “screening” (e.g., the R-locus trait). Of course, manyexamples of suitable marker genes or reporter genes are known to the artand can be employed in the practice of the invention.

[0114] Included within the terms selectable or screenable marker genesare also genes that encode a “secretable marker” whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA, and proteins that are inserted or trapped in the cellwall (e.g., proteins that include a leader sequence such as that foundin the expression unit of extensin or tobacco PR-S).

[0115] With regard to selectable secretable markers, the use of a genethat encodes a protein that becomes sequestered in the cell wall, andwhich protein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

[0116] One example of a protein suitable for modification in this manneris extensin, or hydroxyproline rich glycoprotein (HPRG). The use of theHPRG (Stiefel et al., 1990) is preferred as this molecule is wellcharacterized in terms of molecular biology, expression, and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., 1989) could be modified bythe addition of an antigenic site to create a screenable marker.

[0117] Elements of the present disclosure are exemplified in detailthrough the use of particular marker genes. However in light of thisdisclosure, numerous other possible selectable and/or screenable markergenes will be apparent to those of skill in the art in addition to theone set forth hereinbelow. Therefore, it will be understood that thefollowing discussion is exemplary rather than exhaustive. In light ofthe techniques disclosed herein and the general recombinant techniquesthat are known in the art, the present invention renders possible theintroduction of any gene, including marker genes, into a recipient cellto generate a transformed monocot.

[0118] a. Selectable Markers

[0119] Possible selectable markers for use in connection with thepresent invention include, but are not limited to, a neo gene (Potrykuset al., 1985) that codes for kanamycin resistance and can be selectedfor using kanamycin, G418, and the like; a bar gene that codes forbialaphos resistance; a gene that encodes an altered EPSP synthaseprotein (Hinchee et al., 1988) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae that confersresistance to bromoxynil (Stalker et al., 1988); a mutant acetolactatesynthase gene (ALS) or acetohydroxyacid synthase gene (AHAS) thatconfers resistance to imidazolinone, sulfonylurea or otherALS-inhibiting chemicals (European Patent Application 154,204); amethotrexate-resistant DHFR gene (Thillet et al., 1988); a dalapondehalogenase gene that confers resistance to the herbicide dalapon (U.S.Pat. No. 5,780,708); or a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan (PCT Publication No. WO97/26366). Where a mutant EPSP synthase gene is employed, additionalbenefit may be realized through the incorporation of a suitablechloroplast transit peptide, CTP (U.S. Pat. No. 4,940,835). See also,Lundquist et al., U.S. Pat. No. 5,508,468.

[0120] An illustrative embodiment of a selectable marker gene capable ofbeing used in systems to select transformants is the genes that encodethe enzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318, which is incorporated byreference herein). The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties that have been reported in transformation of cereals(Potrykus, 1989).

[0121] b. Screenable Markers or Reporter Genes

[0122] Screenable markers that may be employed include, but are notlimited to, a β-glucuronidase or uidA gene (GUS) that encodes an enzymefor which various chromogenic substrates are known; an R-locus gene,which encodes a product that regulates the production of anthocyaninpigments (red color) in plant tissues (Dellaporta et al., 1988); aβ-lactamase gene (Sutcliffe, 1978), which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xyle gene (Zukowsky et al., 1983), which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al.,1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher et al., 1985), which may be employed incalcium-sensitive bioluminescence detection, or a green fluorescentprotein gene (Niedz et al., 1995).

[0123] Genes from the R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex encodes a protein thatacts to regulate the production of anthocyanin pigments in most seed andplant tissue. Maize lines can have one, or as many as four, R allelesthat combine to regulate pigmentation in a developmental and tissuespecific manner. Thus, an R gene introduced into such cells will causethe expression of a red pigment and, if stably incorporated, can bevisually scored as a red sector. If a line carries dominant alleles forgenes encoding the enzymatic intermediates in the anthocyaninbiosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessiveallele at the R locus, transformation of any cell from that line with Rwill result in red pigment formation. Exemplary lines include Wisconsin22 that contains the rg-Stadler allele and TR112, a K55 derivative thatis r-g, b, P1. Alternatively any genotype of maize can be utilized ifthe C1 and R alleles are introduced together.

[0124] It is further proposed that R gene regulatory regions may beemployed in chimeric constructs in order to provide mechanisms forcontrolling the expression of chimeric genes. More diversity ofphenotypic expression is known at the R locus than at any other locus(Coe et al., 1988). It is contemplated that regulatory regions obtainedfrom regions 5′ to the structural R gene would be valuable in directingthe expression of genes, e.g., insect resistance, drought resistance,herbicide tolerance or other protein coding regions. For the purposes ofthe present invention, it is believed that any of the various R genefamily members may be successfully employed (e.g., P, S, Lc, etc.).However, the most preferred will generally be Sn (particularly Sn:bol3).Sn is a dominant member of the R gene complex and is functionallysimilar to the R and B loci in that Sn controls the tissue specificdeposition of anthocyanin pigments in certain seedling and plant cells,therefore, its phenotype is similar to R.

[0125] A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

[0126] 5. Transgenes for Modification

[0127] The present invention provides methods and compositions for thetransformation of plant cells with genes in addition to, or other than,marker genes. Such transgenes will often be genes that direct theexpression of a particular protein or polypeptide product, but they mayalso be DNA segments that are not expressed, e.g., transposons such asDs that do not direct their own tranposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

[0128] The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food or feed content and makeup; physical appearance;male sterility; dry down; standability; prolificacy; starch properties;oil quantity and quality; and the like. One may desire to incorporateone or more genes conferring any such desirable trait or traits, suchas, for example, a gene or genes encoding herbicide resistance.

[0129] In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar or anotherselectable marker gene in combination with either of the above genes. Ofcourse, any two or more transgenes of any description, such as thoseconferring herbicide, insect, disease (viral, bacterial, fungal,nematode) or drought resistance, male sterility, dry down, standability,prolificacy, starch properties, oil quantity and quality, or thoseincreasing yield or nutritional quality may be employed as desired.

[0130] a. Herbicide Resistance

[0131] The DNA segments encoding phosphinothricin acetyltransferase (barand pat), EPSP synthase encoding genes conferring resistance toglyphosate, the glypho sate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme thatinactivates dalapon), herbicide resistant (e.g., sulfonylurea andimidazolinone) acetolactate synthase, and bxn genes (encoding anitrilase enzyme that degrades bromoxynil) are examples of herbicideresistant genes for use in transformation. The bar and pat genes codefor an enzyme, phosphinothricin acetyltransferase (PAT), whichinactivates the herbicide phosphinothricin and prevents this compoundfrom inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate) inplants and most microorganisms. However, genes are known that encodeglyphosate-resistant EPSP synthase enzymes, including mutated EPSPSgenes, e.g., the Salmonella typhimurium aroA CT7 mutant (Comai et al.,1985) and the naturally occurring glyphosate resistant EPSPS fromAgrobacterium, CP4 (U.S. Pat. No. 5,627,061). These genes areparticularly contemplated for use in plant transformation. The deh geneencodes the enzyme dalapon dehalogenase and confers resistance to theherbicide dalapon (U.S. Pat. No. 5,780,708). The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

[0132] b. Insect Resistance

[0133] Potential insect resistance genes that can be introduced includeBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Bt genes may provide resistance to economically importantlepidopteran or coleopteran pests such as European Corn Borer (ECB) andWestern Corn Rootworm, respectively. It is contemplated that preferredBt genes for use in the transformation protocols disclosed herein willbe those in which the coding sequence has been modified to effectincreased expression in plants, and more particularly, in maize. Meansfor preparing synthetic genes are well known in the art and aredisclosed in, for example, U.S. Pat. No. 5,500,365 and U.S. Pat. No.5,689,052, each of the disclosures of which are specificallyincorporated herein by reference in their entirety. Examples of suchmodified Bt toxin genes include a synthetic Bt CryIA(b) gene (Perlak etal., 1991), and the synthetic CryIAC gene termed 1800b (U.S. Pat. No.5,590,390). Some examples of other Bt toxin genes known to those ofskill in the art are given in Table 2 below. TABLE 2 Bacillusthuringiensis δ-Endotoxin Genes^(a) New Nomenclature Old NomenclatureGenBank Accession Cry1Aa CryIA(a) M11250 Cry1Ab CryIA(b) M13898 Cry1AcCryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1BaCryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1DbPrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIFM63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725 Cry1HaPrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV U07642 Cry1JaET4 L32019 Cry1Ib ET1 U31527 Cry1K U28801 Cry2Aa CRyIIA M31738 Cry2AbCryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIIBX17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry6ACryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICbU04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8C CryIIIF U04366Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry10A CryIVCM12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12A CryVB L07027Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDa M76442 Cry16A cbm71X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049 Cry19A Jeg65 Y08920Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytB Z14147 Cyt2B CytBU52043

[0134] Protease inhibitors also may provide insect resistance (Johnsonet al., 1989), and thus will have utility in plant transformation. Theuse of a protease inhibitor II gene, pinII, from tomato or potato isenvisioned to be particularly useful. Even more advantageous is the useof a pinII gene in combination with a Bt toxin gene, the combined effectof which has been discovered to produce synergistic insecticidalactivity. Other genes that encode inhibitors of the insect's digestivesystem, or those that encode enzymes or co-factors that facilitate theproduction of inhibitors, also may be useful. This group may beexemplified by oryzacystatin and amylase inhibitors such as those fromwheat and barley.

[0135] Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins that have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et a., 1984),with WGA being preferred.

[0136] Genes controlling the production of large or small polypeptidesactive against insects when introduced into the insect pests, such as,e.g., lytic peptides, peptide hormones and toxins and venoms, formanother aspect of the invention. For example, it is contemplated thatthe expression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

[0137] Transgenic plants expressing genes that encode enzymes thataffect the integrity of the insect cuticle form yet another aspect ofthe invention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant plants. Genes thatcode for activities that affect insect molting, such as those affectingthe production of ecdysteroid UDP-glucosyl transferase, also fall withinthe scope of the useful transgenes of the present invention.

[0138] Genes that code for enzymes that facilitate the production ofcompounds that reduce the nutritional quality of the host plant toinsect pests also are encompassed by the present invention. It may bepossible, for instance, to confer insecticidal activity on a plant byaltering its sterol composition. Sterols are obtained by insects fromtheir diet and are used for hormone synthesis and membrane stability.Therefore, alterations in plant sterol composition by expression ofnovel genes, e.g., those that directly promote the production ofundesirable sterols or those that convert desirable sterols intoundesirable forms, could have a negative effect on insect growth and/ordevelopment and hence endow the plant with insecticidal activity.Lipoxygenases are naturally occurring plant enzymes that have been shownto exhibit anti-nutritional effects on insects and to reduce thenutritional quality of their diet. Therefore, further embodiments of theinvention concern transgenic plants with enhanced lipoxygenase activitythat may be resistant to insect feeding.

[0139]Tripsacum dactyloides is a species of grass that is resistant tocertain insects, including corn root wonn. It is anticipated that genesencoding proteins that are toxic to insects or are involved in thebiosynthesis of compounds toxic to insects will be isolated fromTripsacum and that these novel genes will be useful in conferringresistance to insects. It is known that the basis of insect resistancein Tripsacum is genetic, because said resistance has been transferred toZea mays via sexual crosses (Branson and Guss, 1972). It further isanticipated that other cereal, monocot or dicot plant species may havegenes encoding proteins that are toxic to insects that would be usefulfor producing insect resistant plants.

[0140] Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987), which may be used as a rootworn deterrent;genes encoding avermectin (Avermectin and Abamectin., Campbell, 1989;Ikeda et al., 1987), which may prove particularly useful as a cornrootworm deterrent; ribosome inactivating protein genes; and even genesthat regulate plant structures. Transgenic including anti-insectantibody genes and genes that code for enzymes that can convert anon-toxic insecticide (pro-insecticide) applied to the outside of theplant into an insecticide inside the plant also are contemplated.

[0141] c. Environment or Stress Resistance

[0142] Improvement of a plants ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. As the ZMGRP promoterof the instant invention can be induced by such environmental stresses,genes conferring resistance to these conditions may find particular usewith this promoter.

[0143] It is proposed that benefits may be realized in terms ofincreased resistance to freezing temperatures through the introductionof an “antifreeze” protein such as that of the Winter Flounder (Cutleret al., 1989) or synthetic gene derivatives thereof. Improved chillingtolerance also may be conferred through increased expression ofglycerol-3-phosphate acetyltransferase in chloroplasts (Wolter et al.,1992). Resistance to oxidative stress (often exacerbated by conditionssuch as chilling temperatures in combination with high lightintensities) can be conferred by expression of superoxide dismutase(Gupta et al., 1993), and may be improved by glutathione reductase(Bowler et al., 1992). Such strategies may allow for tolerance tofreezing in newly emerged fields as well as extending later maturityhigher yielding varieties to earlier relative maturity zones.

[0144] It is proposed that expression of a gene encoding hemoglobin mayenhance a plant's ability to assimilate and utilize oxygen, resulting inquicker germination, faster growing or maturing crops, or higher cropyields (Holmberg et al. 1997).

[0145] It is contemplated that the expression of novel genes thatfavorably effect plant water content, total water potential, osmoticpotential, and turgor will enhance the ability of the plant to toleratedrought. As used herein, the terms “drought resistance” and “droughttolerance” are used to confer on a plants increased resistance ortolerance to stress induced by a reduction in water availability, ascompared to normal circumstances, and the ability of the plant tofunction and survive in lower-water environments. In this aspect of theinvention it is proposed, for example, that the expression of genesencoding for the biosynthesis of osmotically-active solutes, such aspolyol compounds, may impart protection against drought. Within thisclass are genes encoding for mannitol-L-phosphate dehydrogenase (Lee andSaier, 1983), trehalose-6-phosphate synthase (Kaasen et al., 1992), andmyo-inositol O-methyl transferase (U.S. Pat. No. 5,563,324). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., 1992,1993). Altered water utilization in transgenic corn producing mannitolalso has been demonstrated (U.S. Pat. No. 5,780,709).

[0146] Similarly, the efficacy of other metabolites in protecting eitherenzyme function (e.g., alanopine or propionic acid) or membraneintegrity (e.g., alanopine) has been documented (Loomis et al., 1989),and therefore expression of genes encoding for the biosynthesis of thesecompounds might confer drought resistance in a manner similar to orcomplimentary to mannitol. Other examples of naturally occurringmetabolites that are osmotically active and/or provide some directprotective effect during drought and/or desiccation include fructose,erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al.,1992), glucosylglycerol (Reed et al., 1984; Erdmann et al., 1992),sucrose, stachyose (Koster and Leopold, 1988; Blackman et al., 1992),raffinose (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al.,1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes that promote thesynthesis of an osmotically active polyol compound are genes that encodethe enzymes mannitol-1-phosphate dehydrogenase, trehalose-6-phosphatesynthase and myoinositol 0-methyltransferase.

[0147] It is contemplated that the expression of specific proteins alsomay increase drought tolerance. Three classes of late embryogenicproteins (LEP) have been assigned based on structural similarities (seeDure et al., 1989). All three classes of LEAs have been demonstrated inmaturing (i.e., desiccating) seeds. Within these 3 types of LEAproteins, the Type-II (dehydrin-type) have generally been implicated indrought and/or desiccation tolerance in vegetative plant parts (i.e.,Mundy and Chua, 1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki etal., 1992). Recently, expression of a Type-III LEA (HVA-1) in tobaccowas found to influence plant height, maturity and drought tolerance(Fitzpatrick, 1993). In rice, expression of the HVA-1 gene influencedtolerance to water deficit and salinity (Xu et al., 1996). Expression ofstructural genes from all three LEA groups may therefore confer droughttolerance. Other types of proteins induced during water stress includethiol proteases, aldolases and transmembrane transporters (Guerrero etal., 1990), which may confer various protective and/or repair-typefunctions during drought stress. It also is contemplated that genes thateffect lipid biosynthesis and hence membrane composition might also beuseful in conferring drought resistance on the plant.

[0148] Many of these genes for improving drought resistance havecomplementary modes of action. Thus, it is envisaged that combinationsof these genes might have additive and/or synergistic effects inimproving drought resistance in crop plants such as, for example, corn,soybean, cotton, or wheat. Many of these genes also improve freezingtolerance (or resistance); the physical stresses incurred duringfreezing and drought are similar in nature and may be mitigated insimilar fashion. Benefit may be conferred via constitutive expression ofthese genes, but the preferred means of expressing these novel genes maybe through the use of a turgor-induced promoter (such as the promotersfor the turgor-induced genes described in Guerrero et al., 1990 andShagan et al., 1993, which are incorporated herein by referenceInducible, spatial and temporal expression patterns of these genes mayenable plants to better withstand stress.

[0149] It is proposed that expression of genes that are involved withspecific morphological traits that allow for increased water extractionsfrom drying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

[0150] Given the overall role of water in determining yield, it iscontemplated that enabling corn and other crop plants to utilize watermore efficiently, through the introduction and expression of novelgenes, will improve overall performance even when soil wateravailability is not limiting. By introducing genes that improve theability of plants to maximize water usage across a full range ofstresses relating to water availability, yield stability or consistencyof yield performance may be realized.

[0151] d. Disease Resistance

[0152] It is proposed that increased resistance to diseases may berealized through introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, bacteria, fungi and nematodes.It also is contemplated that control of mycotoxin producing organismsmay be realized through expression of introduced genes.

[0153] Resistance to viruses may be produced through expression of novelgenes. For example, it has been demonstrated that expression of a viralcoat protein in a transgenic plant can impart resistance to infection ofthe plant by that virus and perhaps other closely related viruses(Cuozzo et al., 1988, Hemenway et al., 1988, Abel et al., 1986). It iscontemplated that expression of antisense genes targeted at essentialviral functions also may impart resistance to viruses. For example, anantisense gene targeted at the gene responsible for replication of viralnucleic acid may inhibit replication and lead to resistance to thevirus. It is believed that interference with other viral functionsthrough the use of antisense genes also may increase resistance toviruses. Similarly, ribozyrnes could be used in this context. Further,it is proposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses. Examples of viral and viral-like diseases, for whichone could introduce resistance to in a transgenic plant in accordancewith the instant invention, are listed below, in Table 3. TABLE 3 PlantVirus and Virus-like Diseases DISEASE CAUSATIVE AGENT American wheatstriate (wheat American wheat striate mosaic striate mosaic) virusmosaic (AWSMV) Barley stripe mosaic Barley stripe mosaic virus (BSMV)Barley yellow dwarf Barley yellow dwarf virus (BYDV) Brome mosaic Bromemosaic virus (BMV) Cereal chlorotic mottle* Cereal chlorotic mottlevirus (CCMV) Corn chlorotic vein banding Corn chlorotic vein bandingvirus (Brazilian mosaic)¹ (CCVBV) Corn lethal necrosis Virus complex(chlorotic mottle virus (MCMV) and dwarf mosaic virus (MDMV) A or B orWheat streak mosaic virus (WSMV)) Cucumber mosaic Cucumber mosaic virus(CMV) Cynodon chlorotic streak*^(,1) Cynodon chlorotic streak virus(CCSV) Johnsongrass mosaic Johnsongrass mosaic virus (JGMV) bushy stuntMycoplasma-like organism (MLO) associated chlorotic dwarf chloroticdwarf virus (MCDV) chlorotic mottle chlorotic mottle virus (MCMV) dwarfmosaic dwarf mosaic virus (MDMV) strains A, D, E and F leaf fleck leaffleck virus (MLFV) line* line virus (MLV) mosaic (corn leaf stripe,mosaic virus (MMV) enanismo rayado) mottle and chlorotic stunt¹ mottleand chlorotic stunt virus* pellucid ringspot* pellucid ringspot virus(MPRV) raya gruesa*^(,1) raya gruesa virus (MRGV) rayado fino* (finestriping rayado fino virus (MRFV) disease) red leaf and red stripe*Mollicute red stripe* red stripe virus (MRSV) ring mottle* ring mottlevirus (MRMV) rio IV* rio cuarto virus (MRCV) rough dwarf* (nanismoruvido) rough dwarf virus (MRDV) (= Cereal tillering disease virus*)sterile stunt* sterile stunt virus (strains of barley yellow striatevirus) streak* streak virus (MSV) stripe (chlorotic stripe, hoja stripevirus blanca) stunting*^(,1) Stunting virus tassel abortion* tasselabortion virus (MTAV) vein enation* vein enation virus (MVEV) wallabyear* wallaby ear virus (MWEV) white leaf* white leaf virus white linemosaic white line mosaic virus (MWLMV) Millet red leaf* Millet red leafvirus (MRLV) Northern cereal mosaic* Northern cereal mosaic virus (NCMV)Oat pseudorosette* Oat pseudorosette virus (zakuklivanie) Oat steriledwarf* Oat sterile dwarf virus (OSDV) Rice black-streaked dwarf* Riceblack-streaked dwarf virus (RBSDV) Rice stripe* Rice stripe virus (RSV)Sorghum mosaic Sorghum mosaic virus (SrMV), formerly sugarcane mosaicvirus (SCMV) strains H, I and M Sugarcane Fiji disease* Sugarcane Fijidisease virus (FDV) Sugarcane mosaic Sugarcane mosaic virus (SCMV)strains A, B, D, E, SC, BC, Sabi and MB (formerly MDMV-B) Veinenation*^(,1) Virus? Wheat spot mosaic¹ Wheat spot mosaic virus (WSMV)

[0154] It is proposed that increased resistance to diseases caused bybacteria and fungi also may be realized through introduction of novelgenes. It is contemplated that genes encoding so-called “peptideantibiotics,” pathogenesis related (PR) proteins, toxin resistance, andproteins affecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences that are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants may be useful in conferring resistance to bacterial disease.These genes are induced following pathogen attack on a host plant andhave been divided into at least five classes of proteins (Bol et al.,1990). Included amongst the PR proteins are β-1,3-glucanases,chitinases, and osmotin and other proteins that are believed to functionin plant resistance to disease organisms. Other genes have beenidentified that have antiflimgal properties, e.g., UDA (stinging nettlelectin), hevein (Broakaert et al., 1989; Barkai-Golan et al., 1978), andsor1 conferring resistance to photosensitizing toxins (Ehrenshaft etal., 1999). It is known that certain plant diseases are caused by theproduction of phytotoxins. It is proposed that resistance to thesediseases would be achieved through expression of a novel gene thatencodes an enzyme capable of degrading or otherwise inactivating thephytotoxin. It also is contemplated that expression of novel genes thatalter the interactions between the host plant and pathogen may be usefulin reducing the ability of the disease organism to invade the tissues ofthe host plant, e.g., an increase in the waxiness of the leaf cuticle orother morphological characteristics. Examples of bacterial and fungaldiseases, including downy mildews, for which one could introduceresistance to in a transgenic plant in accordance with the instantinvention, are listed below, in Tables 4, 5 and 6. TABLE 4 PlantBacterial Diseases DISEASE CAUSATIVE AGENT Bacterial leaf blight andstalk rot Pseudomonas avenae subsp. avenae Bacterial leaf spotXanthomonas campestris pv. holcicola Bacterial stalk rot Enterobacterdissolvens = Erwinia dissolvens Bacterial stalk and top rot Erwiniacarotovora subsp. carotovora, Erwinia chrysanthemi pv. zeae Bacterialstripe Pseudomonas andropogonis Chocolate spot Pseudomonas syringae pv.coronafaciens Goss's bacterial wilt and blight (leaf Clavibactermichiganensis freckles and wilt) subsp. nebraskensis = Corynebacteriummichiganense pv. nebraskense Holcus spot Pseudomonas syringae pv.syringae Purple leaf sheath Hemiparasitic bacteria + (See under Fungi)Seed rot-seedling blight Bacillus subtilis Stewart's disease (bacterialwilt) Pantoea stewartii = Erwinia stewartii Corn stunt(achapparramiento, stunt, Spiroplasma kunkelii Mesa Central or RioGrande stunt)

[0155] TABLE 5 Plant Fungal Diseases DISEASE PATHOGEN Anthracnose leafblight and Colletotrichum graminicola anthracnose stalk rot (teleomorph:Glomerella graminicola Politis), Glomerella tucumanensis (anamorph:Glomerella falcatum Went) Aspergillus ear and kernel rot Aspergillusflavus Link:Fr. Banded leaf and sheath spot* Rhizoctonia solani Kühn =Rhizoctonia microsclerotia J. Matz (teleomorph: Thanatephorus cucumeris)Black bundle disease Acremonium strictum W. Gams = Cephalosporiumacremonium Auct. non Corda Black kernel rot* Lasiodiplodia theobromae =Botryodiplodia theobromae Borde blanco* Marasmiellus sp. Brown spot(black spot, stalk rot) Physoderma maydis Cephalosporium kernel rotAcremonium strictum = Cephalosporium acremonium Charcoal rotMacrophomina phaseolina Corticium ear rot* Thanatephorus cucumeris =Corticium sasakii Curvularia leaf spot Curvularia clavata, C.eragrostidis, = C. maculans (teleomorph: Cochliobolus eragrostidis),Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolusintermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus),Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvulariasenegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)Didymella leaf spot* Didymella exitalis Diplodia ear rot and stalk rotDiplodia frumenti (teleomorph: Botryosphaeria festucae) Diplodia earrot, stalk rot, seed rot and Diplodia maydis = Stenocarpella seedlingblight maydis Diplodia leaf spot or leaf streak Stenocarpella macrospora= Diplodia macrospora

[0156] TABLE 6 Plant Downy Mildews DISEASE CAUSATIVE AGENT Brown stripedowny mildew* Sclerophthora rayssiae var. zeae Crazy top downy mildewSclerophthora macrospora = Sclerospora macrospora Green ear downy mildew(graminicola Sclerospora graminicola downy mildew) Java downy mildew*Peronosclerospora maydis = Sclerospora maydis Philippine downy mildew*Peronosclerospora philippinensis = Sclerospora philippinensis Sorghumdowny mildew Peronosclerospora sorghi = Sclerospora sorghi Spontaneumdowny mildew* Peronosclerospora spontanea = Sclerospora spontaneaSugarcane downy mildew* Peronosclerospora sacchari = Sclerosporasacchari Dry ear rot (cob, kernel and stalk rot) Nigrospora oryzae(teleomorph: Khuskia oryzae) Ear rots, minor Alternaria alternata = A.tenuis, Aspergillus glaucus, A. niger, Aspergillus spp., Botrytiscinerea (teleomorph: Botryotinia fuckeliana), Cunninghamella sp.,Curvularia pallescens, Doratomyces stemonitis = Cephalotrichumstemonitis, Fusarium culmorum, Gonatobotrys simplex, Pithomycesmaydicus, Rhizopus microsporus Tiegh., R. stolonifer = R. nigricans,Scopulariopsis brumptii. Ergot* (horse's tooth, diente de Clavicepsgigantea caballo) (anamorph: Sphacelia sp.) Eyespot Aureobasidium zeae =Kabatiella zeae Fusarium ear and stalk rot Fusarium subglutinans = F.moniliforme var. subglutinans Fusarium kernel, root and stalk rot,Fusarium moniliforme seed rot and seedling blight (teleomorph:Gibberella fujikuroi) Fusarium stalk rot, seedling root rot Fusariumavenaceum (teleomorph: Gibberella avenacea) Gibberella ear and stalk rotGibberella zeae (anamorph: Fusarium graminearum) Gray ear rotBotryosphaeria zeae = Physalospora zeae (anamorph: Macrophoma zeae) Grayleaf spot (Cercospora leaf spot) Cercospora sorghi = C. sorghi var.maydis, C. zeae-maydis Helminthosporium root rot Exserohilumpedicellatum = Helminthosporium pedicellatum (teleomorph: Setosphaeriapedicellata) Hormodendrum ear rot (Cladosporium Cladosporiumcladosporioides = rot) Hormodendrum cladosporioides, C. herbarum(teleomorph: Mycosphaerella tassiana) Hyalothyridium leaf spot*Hyalothyridium maydis Late wilt* Cephalosporium maydis Leaf spots, minorAlternaria alternata, Ascochyta maydis, A. tritici, A. zeicola,Bipolaris victoriae = Helminthosporium victoriae (teleomorph:Cochliobolus victoriae), C. sativus (anamorph: Bipolaris sorokiniana =H. sorokinianum = H. sativum), Epicoccum nigrum, Exserohilum prolatum =Drechslera prolata (teleomorph: Setosphaeria prolata) Graphiumpenicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerellaherpotricha, (anamorph: Scolecosporiella sp.), Paraphaeosphaeriamichotii, Phoma sp., Septoria zeae, S. zeicola, S. zeina Northern cornleaf blight (white blast, Setosphaeria turcica (anamorph: crown stalkrot, stripe) Exserohilum turcicum = Helminthosporium turcicum) Northerncorn leaf spot, Cochliobolus carbonum Helminthosporium ear rot (race 1)(anamorph: Bipolaris zeicola = Helminthosporium carbonum) Penicilliumear rot (blue eye, blue Penicillium spp., P. mold) chrysogenum, P.expansum, P. oxalicum Phaeocytostroma stalk rot and root rotPhaeocytostroma ambiguum, Phaeocytosporella zeae Phaeosphaeria leafspot* Phaeosphaeria maydis Sphaerulina maydis Physalospora ear rot(Botryosphaeria Botryosphaeria festucae = ear rot) Physalospora zeicola(anamorph: Diplodia frumenti) Purple leaf sheath Hemiparasitic bacteriaand fungi Pyrenochaeta stalk rot and root rot Phoma terrestris =Pyrenochaeta terrestris Pythium root rot Pythium spp., P. arrhenomanes,P. graminicola Pythium stalk rot Pythium aphanidermatum = P. butleri L.Red kernel disease (ear mold, leaf and Epicoccum nigrum seed rot)Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph:Waitea circinata) Rhizoctonia root rot and stalk rot Rhizoctonia solani,Rhizoctonia zeae Root rots, minor Alternaria alternata, Cercosporasorghi, Dictochaeta fertilis, Fusarium acuminatum (teleomorph:Gibberella acuminata), F. equiseti (teleomorph: G. intricans), F.oxysporum, F. pallidoroseum, F. poae, F. roseum, G. cyanogena,(anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., Periconiacircinata, Phytophthora cactorum, P. drechsleri, P. nicotianae var.parasitica, Rhizopus arrhizus Rostratum leaf spot Setosphaeria rostrata,(Helminthosporium leaf disease, ear (anamorph: Exserohilum and stalkrot) rostratum = Helminthosporium rostratum) Rust, common corn Pucciniasorghi Rust, southern corn Puccinia polysora Rust, tropical cornPhysopella pallescens, P. zeae = Angiopsora zeae Sclerotium ear rot*(southern blight) Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii)Seed rot-seedling blight Bipolaris sorokiniana, B. zeicola =Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum,Exserohilum turcicum = Helminthosporium turcicum, Fusarium avenaceum, F.culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum),Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp.,Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.Selenophoma leaf spot* Selenophoma sp. Sheath rot Gaeumannomycesgraminis Shuck rot Myrothecium gramineum Silage mold Monascus purpureus,M. ruber Smut, common Ustilago zeae = U. maydis) Smut, falseUstilaginoidea virens Smut, head Sphacelotheca reiliana = Sporisoriumholci-sorghi Southern corn leaf blight and stalk rot Cochliobolusheterostrophus (anamorph: Bipolaris maydis = Helminthosporium maydis)Southern leaf spot Stenocarpella macrospora = Diplodia macrospora Stalkrots, minor Cercospora sorghi, Fusarium episphaeria, F. merismoides, F.oxysporum Schlechtend, F. poae, F. roseum, F. solani (teleomorph:Nectria haematococca), F. tricinctum, Mariannaea elegans, Mucor sp.,Rhopographus zeae, Spicaria sp. Storage rots Aspergillus spp.,Penicillium spp. and other fungi Tar spot* Phyllachora maydisTrichoderma ear rot and root rot Trichoderma viride = T. lignorumteleomorph: Hypocrea sp. White ear rot, root and stalk rot Stenocarpellamaydis = Diplodia zeae Yellow leaf blight Ascochyta ischaemi,Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis) Zonate leafspot Gloeccercospora sorghi

[0157] Plant parasitic nematodes are a cause of disease in many plants,including cereal plants such as maize, barley, wheat, rye and rice. Itis proposed that it would be possible to make plants resistant to theseorganisms through the expression of novel gene products. It isanticipated that control of nematode infestations would be accomplishedby altering the ability of the nematode to recognize or attach to a hostplant and/or enabling the plant to produce nematicidal compounds,including but not limited to proteins. It is known that certainendotoxins derived from Bacillus thuringiensis are nematicidal (Bottjeret al., 1985; U.S. Pat. No. 5,831,011). Examples of nematode-associatedplant diseases, for which one could introduce resistance to in atransgenic plant in accordance with the invention are given below, inTable 7. TABLE 7 Parasitic Nematodes DISEASE PATHOGEN Awl Dolichodorusspp., D. heterocephalus Bulb and stem (Europe) Ditylenchus dipsaciBurrowing Radopholus similis Cyst Heterodera avenae, H. zeae, Punctoderachalcoensis Dagger Xiphinema spp., X. americanum, X. mediterraneum Falseroot-knot Nacobbus dorsalis Lance, Columbia Hoplolaimus columbus LanceHoplolaimus spp., H. galeatus Lesion Pratylenchus spp., P. brachyurus,P. crenatus, P. hexincisus, P. neglectus, P. penetrans, P. scribneri, P.thornei, P. zeae Needle Longidorus spp., L. breviannulatus RingCriconemella spp., C. ornata Root-knot Meloidogyne spp., M. chitwoodi,M. incognita, M. javanica Spiral Helicotylenchus spp. Sting Belonolaimusspp., B. longicaudatus Stubby-root Paratrichodorus spp., P. christiei,P. minor, Quinisulcius acutus, Trichodorus spp. Stunt Tylenchorhynchusdubius

[0158] e. Mycotoxin Reduction/Elimination

[0159] Production of mycotoxins, including aflatoxin and fumonisin, byfungi associated with monocotyledonous plants such as cereal plants,including maize, barley, wheat, rye or rice, is a significant factor inrendering the grain not useful. These fungal organisms do not causedisease symptoms and/or interfere with the growth of the plant, but theyproduce chemicals (mycotoxins) that are toxic to animals. It iscontemplated that inhibition of the growth of these fungi would reducethe synthesis of these toxic substances and therefore reduce grainlosses due to mycotoxin contamination. It also is proposed that it maybe possible to introduce novel genes into monocotyledonous plants suchas that would inhibit synthesis of the mycotoxin. Further, it iscontemplated that expression of a novel gene that encodes an enzymecapable of rendering the mycotoxin nontoxic would be useful in order toachieve reduced mycotoxin contamination of grain. The result of any ofthe above mechanisms would be a reduced presence of mycotoxins on grain.

[0160] f. Grain Composition or Quality

[0161] Genes may be introduced into monocotyledonous plants,particularly commercially important cereals, such as maize, barley,wheat, rye or rice, to improve the grain for which the cereal isprimarily grown. A wide range of novel transgenic plants produced inthis manner may be envisioned depending on the particular end use of thegrain.

[0162] The largest use of grain is for feed or food. Introduction ofgenes that alter the composition of the grain may greatly enhance thefeed or food value. The primary components of grain are starch, protein,and oil. Each of these primary components of grain may be improved byaltering its level or composition. Several examples may be mentioned forillustrative purposes, but in no way provide an exhaustive list ofpossibilities.

[0163] The protein of cereal grains including maize, barley, wheat, ryeand rice is suboptimal for feed and food purposes especially when fed tomonogastric animals such as pigs, poultry, and humans. The protein isdeficient in several amino acids that are essential in the diet of thesespecies, requiring the addition of supplements to the grain. Limitingessential amino acids may include lysine, methionine, tryptophan,threonine, valine, arginine, and histidine. Some amino acids becomelimiting only after corn is supplemented with other inputs for feedformulations. For example, when corn is supplemented with soybean mealto meet lysine requirements methionine becomes limiting. The levels ofthese essential amino acids in seeds and grain may be elevated bymechanisms that include, but are not limited to, the introduction ofgenes to increase the biosynthesis of the amino acids, decrease thedegradation of the amino acids, increase the storage of the amino acidsin proteins, direct the storage of amino acids in proteins comprising anutritionally enhanced balance of amino acids, or increase transport ofthe amino acids to the seeds or grain.

[0164] One mechanism for increasing the biosynthesis of the amino acidsis to introduce genes that deregulate the amino acid biosyntheticpathways such that the plant can no longer adequately control the levelsthat are produced. This may be done by deregulating or bypassing stepsin the amino acid biosynthetic pathway that are normally regulated bylevels of the amino acid end product of the pathway. Examples includethe introduction of genes that encode deregulated versions of theenzymes aspartokinase or dihydrodipicolinic acid (DHDP)-synthase forincreasing lysine and threonine production, and anthranilate synthasefor increasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyze steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase. It is anticipated that it may bedesirable to target expression of genes relating to amino acidbiosynthesis to the endosperm or embryo of the seed. More preferably,the gene will be targeted to the embryo. It will also be preferable forgenes encoding proteins involved in amino acid biosynthesis to targetthe protein to a plastid using a plastid transit peptide sequence.

[0165] The protein composition of the grain may be altered to improvethe balance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.Examples may include the introduction of DNA that decreases theexpression of members of the zein family of storage proteins. This DNAmay encode ribozymes or antisense sequences directed to impairingexpression of zein proteins or expression of regulators of zeinexpression such as the opaque-2 gene product. It also is proposed thatthe protein composition of the grain may be modified through thephenomenon of co-suppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al., 1991;PCT Publication No. WO 98/26064). Additionally, the introduced DNA mayencode enzymes that degrade zeins. The decreases in zein expression thatare achieved may be accompanied by increases in proteins with moredesirable amino acid composition or increases in other major seedconstituents such as starch. Alternatively, a chimeric gene may beintroduced that comprises a coding sequence for a native protein ofadequate amino acid composition such as for one of the globulin proteinsor 10 kD delta zein or 20 kD delta zein or 27 kD gamma zein of and apromoter or other regulatory sequence designed to elevate expression ofsaid protein. The coding sequence of the gene may include additional orreplacement codons for essential amino acids. Further, a coding sequenceobtained from another species, or, a partially or completely syntheticsequence encoding a completely unique peptide sequence designed toenhance the amino acid composition of the seed may be employed. It isanticipated that it may be preferable to target expression of thesetransgenes encoding proteins with superior composition to the endospermof the seed.

[0166] The introduction of genes that alter the oil content of the grainmay be of value. Increases in oil content may result in increases inmetabolizable-energy-content and density of the seeds for use in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, β-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feed stuff. The introduced DNA also mayencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in thegrain such as described below. Some other examples of genes specificallycontemplated by the inventors for use in creating transgenic plants withaltered oil composition traits include 2-acetyltransferase, oleosin,pyruvate dehydrogenase complex, acetyl CoA synthetase, ATP citratelyase, ADP-glucose pyrophosphorylase and genes of thecarnitine-CoA-acetyl-CoA shuttles. It is anticipated that expression ofgenes related to oil biosynthesis will be targeted to the plastid, usinga plastid transit peptide sequence and preferably expressed in the seedembryo.

[0167] Genes may be introduced that enhance the nutritive value of thestarch component of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch, for example,in cows by delaying its metabolism. It is contemplated that alterationof starch structure may improve the wet milling properties of grain ormay produce a starch composition with improved qualities for industrialutilization. It is anticipated that expression of genes related tostarch biosynthesis will preferably be targeted to the endosperm of theseed.

[0168] Besides affecting the major constituents of the grain, genes maybe introduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA that blocks or eliminates steps in pigment production pathways.

[0169] Most of the phosphorous content of the grain is in the form ofphytate, a form of phosphate storage that is not metabolized bymonogastric animals. Therefore, in order to increase the availability ofseed phosphate, it is anticipated that one will desire to decrease theamount of phytate in seed and increase the amount of free phosphorous.It is anticipated that one can decrease the expression or activity ofone of the enzymes involved in the synthesis of phytate. For example,suppression of expression of the gene encoding inositol phosphatesynthetase (INOPS) may lead to an overall reduction in phytateaccumulation. It is anticipated that antisense or sense suppression ofgene expression may be used. Alternatively, one may express a gene inseed that will be activated, e.g., by pH, in the gastric system of amonogastric animal and will release phosphate from phytate, e.g.,phytase. It is further contemplated that one may provide an alternatestorage form for phosphate in the grain, wherein the storage form ismore readily utilized by a monogastric animal.

[0170] Feed or food comprising primarily maize or other cereal grainspossesses insufficient quantities of vitamins and must be supplementedto provide adequate nutritive value. Introduction of genes that enhancevitamin biosynthesis in seeds may be envisioned including, for example,vitamins A, E, B₁₂, choline, and the like. Maize grain also does notpossess sufficient mineral content for optimal nutritive value. Genesthat affect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase thatenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

[0171] Numerous other examples of improvement of other plants for feedand food purposes might be described. The improvements may not evennecessarily involve the grain, but may, for example, improve the valueof the plants for silage. Introduction of DNA to accomplish this mightinclude sequences that alter lignin production such as those that resultin the “brown midrib” phenotype associated with superior feed value forcattle.

[0172] In addition to direct improvements in feed or food value, genesalso may be introduced that improve the processing of plant material andimprove the value of the products resulting from the processing. Forexample, the primary method of processing maize is via wetmilling thatmay be improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

[0173] Improving the value of wetmilling products may include alteringthe quantity or quality of starch, oil, corn gluten meal, or thecomponents of corn gluten feed. Elevation of starch may be achievedthrough the identification and elimination of rate limiting steps instarch biosynthesis or by decreasing levels of the other components ofthe grain resulting in proportional increases in starch. An example ofthe former may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or that areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

[0174] The properties of starch may be beneficially altered by changingthe ratio of amylose to amylopectin, the size of the starch molecules,or their branching pattern. Through these changes a broad range ofproperties may be modified that include, but are not limited to, changesin gelatinization temperature, heat of gelatinization, clarity of filmsand pastes, rheological properties, and the like. To accomplish thesechanges in properties, genes that encode granule-bound or soluble starchsynthase activity or branching enzyme activity may be introduced aloneor combination. DNA such as antisense constructs also may be used todecrease levels of endogenous activity of these enzymes. The introducedgenes or constructs may possess regulatory sequences that time theirexpression to specific intervals in starch biosynthesis and starchgranule development. Furthermore, it may be worthwhile to introduce andexpress genes that result in the in vivo derivatization, or othermodification, of the glucose moieties of the starch molecule. Thecovalent attachment of any molecule may be envisioned, limited only bythe existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups that provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

[0175] Oil is another product of wetmilling of corn, the value of whichmay be improved by introduction and expression of genes. The quantity ofoil that can be extracted by wetmilling may be elevated by approaches asdescribed for feed and food above. Oil properties also may be altered toimprove its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized that upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids and the lipids possessing them or by increasing levelsof native fatty acids while possibly reducing levels of precursors.Alternatively, DNA sequences may be introduced that slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, and other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid and oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C₈ toC₁₂ saturated fatty acids.

[0176] Improvements in the other major corn wetmilling products, corngluten meal and corn gluten feed, also may be achieved by theintroduction of genes to obtain novel corn plants. Representativepossibilities include but are not limited to those described above forimprovement of food and feed value.

[0177] In addition, it may further be considered that a plant, such asmaize or other monocots, may be used for the production or manufacturingof useful biological compounds that were either not produced at all, ornot produced at the same level, in the plant previously. The novelplants producing these compounds are made possible by the introductionand expression of genes by transformation methods. The vast array ofpossibilities include but are not limited to any biological compoundthat is presently produced by any organism such as proteins, nucleicacids, primary and intermediary metabolites, carbohydrate polymers, etc.The compounds may be produced by the plant, extracted upon harvestand/or processing, and used for any presently recognized useful purposesuch as pharmaceuticals, fragrances, and industrial enzymes to name afew.

[0178] Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance y-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes that affect flavor such as theshrunken 1 gene (encoding sucrose synthase) or shrunken 2 gene (encodingADPG pyrophosphorylase) for sweet corn.

[0179] g. Plant Agronomic Characteristics

[0180] Two of the factors determining where crop plants can be grown arethe average daily temperature during the growing season and the lengthof time between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time thecrop has available to grow to maturity and be harvested. For example, tobe grown in a particular area is selected for its ability to mature anddry down to harvestable moisture content within the required period oftime with maximum possible yield. Therefore, plants, including maize orother cereals, of varying maturities are developed for different growinglocations. Apart from the need to dry down sufficiently to permitharvest, it is desirable to have maximal drying take place in the fieldto minimize the amount of energy required for additional post-harvestdrying. Also, the more readily the grain can dry down, the more timethere is available for growth and seed maturation. It is considered thatgenes that influence maturity and/or dry down can be identified andintroduced into corn or other plants using transformation techniques tocreate new varieties adapted to different growing locations or the samegrowing location, but having improved yield to moisture ratio atharvest. Expression of genes that are involved in regulation of plantdevelopment may be especially useful, e.g., the liguleless and roughsheath genes that have been identified in corn.

[0181] It is contemplated that genes may be introduced into plants thatwould improve standability and other plant growth characteristics.Expression of novel genes that confer stronger stalks, improved rootsystems, or prevent or reduce ear droppage would be of great value tothe farmer. It is proposed that introduction and expression of genesthat increase the total amount of photoassimilate available by, forexample, increasing light distribution and/or interception would beadvantageous. In addition, the expression of genes that increase theefficiency of photosynthesis and/or the leaf canopy would furtherincrease gains in productivity. It is contemplated that expression of aphytochrome gene in plants, including maize, may be advantageous.Expression of such a gene may reduce apical dominance, confersemidwarfism on a plant, and increase shade tolerance (U.S. Pat. No.5,268,526). Such approaches would allow for increased plant populationsin the field.

[0182] Delay of late season vegetative senescence would increase theflow of assimilate into the grain and thus increase yield. It isproposed that overexpression of genes within a plant such as maize thatare associated with “stay green” or the expression of any gene thatdelays senescence would be advantageous. For example, a nonyellowingmutant has been identified in Festuca pratensis (Davies et al., 1990).Expression of this gene as well as others may prevent prematurebreakdown of chlorophyll and thus maintain canopy function.

[0183] h. Nutrient Utilization

[0184] The ability to utilize available nutrients may be a limitingfactor in growth of monocotyledonous plants such as maize, barley,wheat, rye or rice. It is proposed that it would be possible to alternutrient uptake, tolerate pH extremes, mobilization through the plant,storage pools, and availability for metabolic activities by theintroduction of novel genes. These modifications would allow a plantsuch as maize, barley, wheat, rye or rice to more efficiently utilizeavailable nutrients. It is contemplated that an increase in the activityof, for example, an enzyme that is normally present in the plant andinvolved in nutrient utilization would increase the availability of anutrient. An example of such an enzyme would be phytase. It further iscontemplated that enhanced nitrogen utilization by a plant is desirable.Expression of a glutamate dehydrogenase gene in plants such as maize,e.g., E. coli gdhA genes, may lead to increased fixation of nitrogen inorganic compounds. Furthermore, expression of gdhA in a plant may leadto enhanced resistance to the herbicide glufosinate by incorporation ofexcess ammonia into glutamate, thereby detoxifying the ammonia. It alsois contemplated that expression of a novel gene may make a nutrientsource available that was previously not accessible, e.g., an enzymethat releases a component of nutrient value from a more complexmolecule, perhaps a macromolecule.

[0185] i. Male Sterility

[0186] Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al., 1990).

[0187] A number of mutations were discovered in maize that confercytoplasmic male sterility. One mutation in particular, referred to as Tcytoplasm, also correlates with sensitivity to Southern corn leafblight. A DNA sequence, designated TURF-13 (Levings, 1990), wasidentified that correlates with T cytoplasm. It is proposed that itwould be possible through the introduction of TURF-13 viatransformation, to separate male sterility from disease sensitivity. Asit is necessary to be able to restore male fertility for breedingpurposes and for grain production, it is proposed that genes encodingrestoration of male fertility also may be introduced.

[0188] j. Negative Selectable Markers

[0189] Introduction of genes encoding traits that can be selectedagainst may be useful for eliminating undesirable linked genes. It iscontemplated that when two or more genes are introduced together bycotransformation that the genes will be linked together on the hostchromosome. For example, a gene encoding Bt that confers insectresistance to the plant may be introduced into a plant together with abar gene that is useful as a selectable marker and confers resistance tothe herbicide LIBERTY® on the plant. However, it may not be desirable tohave an insect resistant plant that also is resistant to the herbicideLIBERTY®. It is proposed that one also could introduce an antisense barcoding region that is expressed in those tissues where one does not wantexpression of the bar gene product, e.g., in whole plant parts. Hence,although the bar gene is expressed and is useful as a selectable marker,it is not useful to confer herbicide resistance on the whole plant. Thebar antisense gene is a negative selectable marker.

[0190] It also is contemplated that negative selection is necessary inorder to screen a population of transformants for rare homologousrecombinants generated through gene targeting. For example, a homologousrecombinant may be identified through the inactivation of a gene thatwas previously expressed in that cell. The antisense construct forneomycin phosphotransferase II (NPT II) has been investigated as anegative selectable marker in tobacco (Nicotiana tabacum) andArabidopsis thaliana (Xiang and Guerra, 1993). In this example, bothsense and antisense NPT II genes are introduced into a plant throughtransformation and the resultant plants are sensitive to the antibiotickanamycin. An introduced gene that integrates into the host cellchromosome at the site of the antisense NPT II gene, and inactivates theantisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare, site-specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

[0191] It is contemplated that negative selectable markers also may beuseful in other ways. One application is to construct transgenic linesin which one could select for transposition to unlinked sites. In theprocess of tagging it is most common for the transposable element tomove to a genetically linked site on the same chromosome. A selectablemarker for recovery of rare plants in which transposition has occurredto an unlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose. In the presence of this enzymethe non-phytotoxic compound 5-fluorocytosine is converted to5-fluorouracil, which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofα-naphthalene acetamide (NAM) to α-naphthalene acetic acid (NAA) whichrenders plant cells sensitive to high concentrations of NAM (Depicker etal., 1988).

[0192] It also is contemplated that negative selectable markers may beuseful in the construction of transposon tagging lines. For example, bymarking an autonomous transposable element such as Ac, Master Mu, orEn/Spn with a negative selectable marker, one could select fortransformants in which the autonomous element is not stably integratedinto the genome. It is proposed that this would be desirable, forexample, when transient expression of the autonomous element is desiredto activate in trans the transposition of a defective transposableelement, such as Ds, but stable integration of the autonomous element isnot desired. The presence of the autonomous element may not be desiredin order to stabilize the defective element, i.e., prevent it fromfurther transposing. However, it is proposed that if stable integrationof an autonomous transposable element is desired in a plant the presenceof a negative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

[0193] It is also contemplated that a negative selectable marker may beuseful for identifying rare homologous recombination events betweendirect repeats. For example, some of the pathways for obtaining adeletion of a transgene (FIG. 1) can occur in hemizygous plant cells,including callus or other regenerative somatic cells during the tissueculture process (Zubko et al, 2000). The recovery of such rare eventsmay be enhanced by screening for the loss (deletion) of a negativeselectable marker gene.

[0194] k. Non-Protein-Expressing Sequences

[0195] DNA may be introduced into plants for the purpose of expressingRNA transcripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of aplant.

[0196] 1. Antisense RNA

[0197] Genes may be constructed or isolated, which when transcribed,produce antisense RNA that is complementary to all or part(s) of atargeted messenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction that include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

[0198] 2. Ribozymes

[0199] Genes also may be constructed or isolated, which whentranscribed, produce RNA enzymes (ribozymes) that can act asendoribonucleases and catalyze the cleavage of RNA molecules withselected sequences. The cleavage of selected messenger RNAs can resultin the reduced production of their encoded polypeptide products. Thesegenes may be used to prepare novel transgenic plants that possess them.The transgenic plants may possess reduced levels of polypeptidesincluding, but not limited to, the polypeptides cited above.

[0200] Ribozymes are RNA-protein complexes that cleave nucleic acids ina site-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (IGS) of the ribozyme priorto chemical reaction.

[0201] Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

[0202] Several different ribozyme motifs have been described with RNAcleavage activity (Symons, 1992). Examples include sequences from theGroup I self-splicing introns including Tobacco Ringspot Virus (Prody etal., 1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons,1981), and Lucerne Transient Streak Virus (Forster and Symons, 1987).Sequences from these and related viruses are referred to as hammerheadribozymes based on a predicted folded secondary structure.

[0203] Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

[0204] The other variable on ribozyme design is the selection of acleavage site on a given target RNA. Ribozymes are targeted to a givensequence by virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence that is the cleavage site. For a hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA, a uracil(U) followed by either an adenine, cytosine or uracil (A, C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

[0205] Designing and testing ribozymes for efficient cleavage of atarget RNA is a process well known to those skilled in the art. Examplesof scientific methods for designing and testing ribozymes are describedby Chowrira et al., (1994) and Lieber and Strauss (1995), eachincorporated by reference. The identification of operative and preferredsequences for use in down regulating a given gene is simply a matter ofpreparing and testing a given sequence, and is a routinely practiced“screening” method known to those of skill in the art.

[0206] 3. Induction of Gene Silencing

[0207] It also is possible that genes may be introduced to produce noveltransgenic plants that have reduced expression of a native gene productby the mechanism of co-suppression. It has been demonstrated in tobacco,tomato, petunia, and corn (Goring et al., 1991; Smith et al., 1990;Napoli et al., 1990; van der Krol et al., 1990; PCT Publication No. WO98/26064) that expression of the sense transcript of a native gene willreduce or eliminate expression of the native gene in a manner similar tothat observed for antisense genes. The introduced gene may encode all orpart of the targeted native protein but its translation may not berequired for reduction of levels of that native protein.

[0208] 4. Non-RNA-Expressing Sequences

[0209] DNA elements including those of transposable elements such as Ds,Ac, or Mu, may be inserted into a gene to cause mutations. These DNAelements may be inserted in order to inactivate (or activate) a gene andthereby “tag” a particular trait. In this instance the transposableelement does not cause instability of the tagged mutation, because theutility of the element does not depend on its ability to move in thegenome. Once a desired trait is tagged, the introduced DNA sequence maybe used to clone the corresponding gene, e.g., using the introduced DNAsequence as a PCR primer target sequence together with PCR gene cloningtechniques (Shapiro, 1983; Dellaporta et al., 1988). Once identified,the entire gene(s) for the particular trait, including control orregulatory regions where desired, may be isolated, cloned andmanipulated as desired. The utility of DNA elements introduced into anorganism for purposes of gene tagging is independent of the DNA sequenceand does not depend on any biological activity of the DNA sequence,i.e., transcription into RNA or translation into protein. The solefunction of the DNA element is to disrupt the DNA sequence of a gene.

[0210] It is contemplated that unexpressed DNA sequences, includingnovel synthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

[0211] Another possible element that may be introduced is a matrixattachment region element (MAR), such as the chicken lysozyme A element(Stief, 1989), which can be positioned around an expressible gene ofinterest to effect an increase in overall expression of the gene anddiminish position dependent effects upon incorporation into the plantgenome (Stief et al., 1989; Phi-Van et al., 1990).

[0212] 5. Other sequences

[0213] An expression cassette of the invention can also be furthercomprise plasmid DNA. Plasmid vectors include additional DNA sequencesthat provide for easy selection, amplification, and transformation ofthe expression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,selectable marker genes, preferably encoding antibiotic or herbicideresistance, unique multiple cloning sites providing for multiple sitesto insert DNA sequences or genes encoded in the expression cassette, andsequences that enhance transformation of prokaryotic and eukaryoticcells.

[0214] Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in U.S. Pat.No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmidvector has been previously characterized by An (1989) and is availablefrom Dr. An. This binary Ti vector can be replicated in prokaryoticbacteria such as E. coli and Agrobacterium. The Agrobacterium plasmidvectors can be used to transfer the expression cassette to plant cells.The binary Ti vectors preferably include the nopaline T DNA right andleft borders to provide for efficient plant cell transformation, aselectable marker gene, unique multiple cloning sites in the T borderregions, the colE1 replication of origin and a wide host range replicon.The binary Ti vectors carrying an expression cassette of the inventioncan be used to transform both prokaryotic and eukaryotic cells, but ispreferably used to transform plant cells.

[0215] In certain embodiments, it is contemplated that one may wish toemploy replication-competent viral vectors in monocot transformation.Such vectors include, for example, wheat dwarf virus (WDV) “shuttle”vectors, such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectorsare capable of autonomous replication in cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs et al., 1990) that transpositionof these elements within the genome requires DNA replication. It is alsocontemplated that transposable elements would be useful for introducingDNA fragments lacking elements necessary for selection and maintenanceof the plasmid vector in bacteria, e.g., antibiotic resistance genes andorigins of DNA replication. It is also proposed that use of atransposable element such as Ac, Ds, or Mu would actively promoteintegration of the desired DNA and hence increase the frequency ofstably transformed cells.

[0216] Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes)and DNA segments for use in transforming such cells will, of course,generally comprise the isolated and purified cDNA(s), isolated andpurified DNA(s) or genes that one desires to introduce into the cells.These DNA constructs can further include structures such as promoters,enhancers, polylinkers, or even regulatory genes as desired. The DNAsegment or gene chosen for cellular introduction will often encode aprotein that will be expressed in the resultant recombinant cells, suchas will result in a screenable or selectable trait and/or that willimpart an improved phenotype to the regenerated plant. However, this maynot always be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

[0217] III. Methods for Plant Transformation

[0218] Suitable methods for plant transformation for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell, such as by direct delivery of DNAsuch as by PEG-mediated transformation of protoplasts (Omirulleh et al.,1993), by desiccation/inhibition-mediated DNA uptake (Potrykus et al.,1985), by electroporation (U.S. Pat. No. 5,384,253, specificallyincorporated herein by reference in its entirety), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523,specifically incorporated herein by reference in its entirety; and U.S.Pat. No. 5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), Through the application of techniques such as these, maizecells as well as those of virtually any other plant species may bestably transformed, and these cells developed into transgenic plants. Incertain embodiments, acceleration methods are preferred and include, forexample, microprojectile bombardment and the like.

[0219] A. Electroporation

[0220] Where one wishes to introduce DNA by means of electroporation, itis contemplated that the method of Krzyzek et al. (U.S. Pat. No.5,384,253, incorporated herein by reference in its entirety) will beparticularly advantageous. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation by mechanical wounding.

[0221] To effect transformation by electroporation, one may employeither friable tissues, such as a suspension culture of cells orembryogenic callus or alternatively one may transform immature embryosor other organized tissue directly. In this technique, one wouldpartially degrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species that have been transformedby electroporation of intact cells include (U.S. Pat. No. 5,384,253;Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993),tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco(Lee et al., 1989).

[0222] One also may employ protoplasts for electroporationtransformation of plants (Bates, 1994; Lazzeri, 1995). For example, thegeneration of transgenic soybean plants by electroporation ofcotyledon-derived protoplasts is described by Dhir and Widholm in PCTPublication No. WO 92/17598 (specifically incorporated herein byreference). Other examples of species for which protoplasttransformation has been described include barley (Lazerri, 1995),sorghum (Battraw et al., 1991), (Bhattacharjee et al., 1997), wheat (Heet al., 1994) and tomato (Tsukada, 1989).

[0223] B. Microprojectile Bombardment

[0224] A preferred method for delivering transforming DNA segments toplant cells in accordance with the invention is microprojectilebombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat.No. 5,610,042; and U.S. Pat. No. 5,590,390; each of which isspecifically incorporated herein by reference in its entirety). In thismethod, particles may be coated with nucleic acids and delivered intocells by a propelling force. Exemplary particles include those comprisedof tungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

[0225] For the bombardment, cells in suspension are concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate.

[0226] An illustrative embodiment of a method for delivering DNA intoplant cells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

[0227] Microprojectile bombardment techniques are widely applicable, andmay be used to transform virtually any plant species. Examples ofspecies for which have been transformed by microprojectile bombardmentinclude monocot species such as maize (U.S. Pat. No. 5,590,390), barley(Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995;Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower etal., 1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as wellas a number of dicots including tobacco (Tomes et al., 1990; Buising andBenbow, 1994), soybean (U.S. Pat. No. 5,322,783, specificallyincorporated herein by reference in its entirety), sunflower (Knittel etal. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell,1993), tomato (Van Eck et al. 1995), and legumes in general (U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety).

[0228] C. Agrobacterium-mediated Transformation

[0229] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

[0230] Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; Zhang et al., 1997; U.S. Pat. No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and rice (Ishida et al., 1996).

[0231] Modem Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0232] Other Transformation Methods

[0233] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

[0234] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts have been described (Toriyama et al., 1986; Yamada et al.,1986; Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No.5,508,184; each specifically incorporated herein by reference in itsentirety). Examples of the use of direct uptake transformation of cerealprotoplasts include transformation of rice (Ghosh-Biswas et al., 1994),sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng andEdwards, 1990) and maize (Omirulleh et al., 1993).

[0235] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1989). Also, silicon carbide fiber-mediated transformation may be usedwith or without protoplasting (Kaeppler, 1990; Kaeppler et al., 1992;U.S. Pat. No. 5,563,055, specifically incorporated herein by referencein its entirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell is punctured. This technique has beenused successfully with, for example, the monocot cereals (U.S. Pat. No.5,590,390, specifically incorporated herein by reference in itsentirety; Thompson, 1995) and rice (Nagatani, 1997).

[0236] IV. Optimization of Microprojectile Bombardment

[0237] For microprojectile bombardment transformation in accordance withthe current invention, both physical and biological parameters may beoptimized. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, such as the osmotic adjustment of targetcells to help alleviate the trauma associated with bombardment, theorientation of an immature embryo or other target tissue relative to theparticle trajectory, and also the nature of the transforming DNA, suchas linearized DNA or intact supercoiled plasmids. It is believed thatpre-bombardment manipulations are especially important for successfultransformation of immature embryos.

[0238] Accordingly, it is contemplated that one may wish to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as DNA concentration, gap distance, flight distance,tissue distance, and helium pressure. It further is contemplated thatthe grade of helium may affect transformation efficiency. For example,differences in transformation efficiencies may be witnessed betweenbombardments using industrial grade (99.99% pure) or ultra pure helium(99.999% pure), although it is not currently clear that is moreadvantageous for use in bombardment. One also may optimize the traumareduction factors (TRFs) by modifying conditions that influence thephysiological state of the recipient cells and that may thereforeinfluence transformation and integration efficiencies. For example, theosmotic state, tissue hydration and the subculture stage or cell cycleof the recipient cells may be adjusted for optimum transformation.

[0239] A. Physical Parameters

[0240] 1. Gap Distance

[0241] The variable nest (macro holder) can be adjusted to vary thedistance between the rupture disk and the macroprojectile, i.e., the gapdistance. This distance can be varied from 0 to 2 cm. The predictedeffects of a shorter gap are an increase of velocity of both the macro-and microprojectiles, an increased shock wave (which leads to tissuesplattering and increased tissue trauma), and deeper penetration ofmicroprojectiles. Longer gap distances would have the opposite effectsbut may increase viability and therefore the total number of recoveredstable transformants.

[0242] 2. Flight Distance

[0243] The fixed nest (contained within the variable nest) can be variedbetween 0.5 and 2.25 cm in predetermined 0.5 cm increments by theplacement of spacer rings to adjust the flight path traversed by themacroprojectile. Short flight paths allow for greater stability of themacroprojectile in flight but reduce the overall velocity of themicroprojectiles. Increased stability in flight increases, for example,the number of centered GUS foci. Greater flight distances (up to apoint) increase velocity but also increase instability in flight. Basedon observations, it is recommended that bombardments typically be donewith a flight path length of about 1.0 cm to 1.5 cm.

[0244] 3. Tissue Distance

[0245] Placement of tissue within the gun chamber can have significanteffects on microprojectile penetration. Increasing the flight path ofthe microprojectiles will decrease velocity and trauma associated withthe shock wave. A decrease in velocity also will result in shallowerpenetration of the microprojectiles.

[0246] 4. Helium Pressure

[0247] By manipulation of the type and number of rupture disks, pressurecan be varied between 400 and 2000 psi within the gas acceleration tube.Optimum pressure for stable transformation has been determined to bebetween 1000 and 1200 psi.

[0248] 5. Coating of Microprojectiles.

[0249] For microprojectile bombardment, one will attach (i.e., “coat”)DNA to the microprojectiles such that it is delivered to recipient cellsin a form suitable for transformation thereof. In this respect, at leastsome of the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of bonds between transforming DNA and the microprojectilefollowing delivery of the microprojectile to the target cell. This neednot be the case, however, as availability to a target cell may occur asa result of breakage of unbound segments of DNA or of other moleculesthat comprise the physical attachment to the microprojectile.Availability may further occur as a result of breakage of bonds betweenthe transforming DNA and other molecules, which are either directly orindirectly attached to the microprojectile. It further is contemplatedthat transformation of a target cell may occur by way of directrecombination between the transforming DNA and the genomic DNA of therecipient cell. Therefore, as used herein, a “coated” microprojectilewill be one that is capable of being used to transform a target cell, inthat the transforming DNA will be delivered to the target cell, yet willbe accessible to the target cell such that transformation may occur.

[0250] Any technique for coating microprojectiles that allows fordelivery of transforming DNA to the target cells may be used. Methodsfor coating microprojectiles that have been demonstrated to work wellwith the current invention have been specifically disclosed herein. DNAmay be bound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend labeled with long chain thiol-cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction, but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

[0251] Alternatively, particles may be prepared by functionalizing thesurface of a gold oxide particle, providing free amine groups. DNA,having a strong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

[0252] As disclosed above, it further is proposed that the concentrationof DNA used to coat microprojectiles may influence the recovery oftransformants containing a single copy of the transgene. For example, alower concentration of DNA may not necessarily change the efficiency ofthe transformation, but may instead increase the proportion of singlecopy insertion events. In this regard, approximately 1 ng to 2000 ng oftransforming DNA may be used per each 1.8 mg of startingmicroprojectiles. In other embodiments of the invention, approximately2.5 ng to 1000 ng, 2.5 ng to 750 ng, 2.5 ng to 500 ng, 2.5 ng to 250 ng,2.5 ng to 100 ng, or 2.5 ng to 50 ng of transforming DNA may be used pereach 1.8 mg of starting microprojectiles.

[0253] Various other methods also may be used to increase transformationefficiency and/or increase the relative proportion of low-copytransformation events. For example, the inventors contemplateend-modifying transforming DNA with alkaline phosphatase or an agentthat will blunt DNA ends prior to transformation. Still further, aninert carrier DNA may be included with the transforming DNA, therebylowering the effective transforming DNA concentration without loweringthe overall amount of DNA used. These techniques are further describedin U.S. patent application Ser. No. 08/995,451, filed Dec. 22, 1997, thedisclosure of which is specifically incorporated herein by reference inits entirety.

[0254] B. Biological Parameters

[0255] Culturing conditions and other factors can influence thephysiological state of the target cells and may have profound effects ontransformation and integration efficiencies. First, the act ofbombardment could stimulate the production of ethylene, which could leadto senescence of the tissue. The addition of antiethylene compoundscould increase transformation efficiencies. Second, it is proposed thatcertain points in the cell cycle may be more appropriate for integrationof introduced DNA. Hence synchronization of cell cultures may enhancethe frequency of production of transformants. For example,synchronization may be achieved using cold treatment, amino acidstarvation, or other cell cycle-arresting agents. Third, the degree oftissue hydration also may contribute to the amount of trauma associatedwith bombardment as well as the ability of the microprojectiles topenetrate cell walls.

[0256] The position and orientation of an embryo or other target tissuerelative to the particle trajectory also may be important. For example,the PDS-1000 Biolistics device does not produce a uniform spread ofparticles over the surface of a target petri dish. The velocity ofparticles in the center of the plate is higher than the particlevelocity at further distances from the center of the petri dish.Therefore, it is advantageous to situate target tissue on the petri dishsuch as to avoid the center of the dish, referred to by some as the“zone of death.” Furthermore, orientation of the target tissue withregard to the trajectory of targets also can be important. It iscontemplated that it is desirable to orient the tissue most likely toregenerate a plant toward the particle stream. For example, thescutellum of an immature embryo comprises the cells of greatestembryogenic potential and therefore should be oriented toward theparticle stream.

[0257] It also has been reported that slightly plasmolyzed yeast cellsallow increased transformation efficiencies (Armaleo et al., 1990). Itwas hypothesized that the altered osmotic state of the cells helped toreduce trauma associated with the penetration of the microprojectile.Additionally, the growth and cell cycle stage may be important withrespect to transformation.

[0258] 1. Osmotic Adjustment

[0259] It has been suggested that osmotic pre-treatment couldpotentially reduce bombardment associated injury as a result of thedecreased turgor pressure of the plasmolyzed cell. In a previous study,the number of cells transiently expressing GUS increased followingsubculture into both fresh medium and osmotically adjusted medium (U.S.Pat. No. 5,590,390, specifically incorporated herein by reference in itsentirety). Pretreatment times of 90 minutes showed higher numbers of GUSexpressing foci than shorter times. Cells incubated in 500 mOSM/kgmedium for 90 minutes showed an approximately 3.5 fold increase intransient GUS foci than the control. Preferably, immature embryos areprecultured for 4-5 hours prior to bombardment on culture mediumcontaining 12% sucrose. A second culture on 12% sucrose is performed for16-24 hours following bombardment. Alternatively, type II cells arepretreated on 0.2M mannitol for 3-4 hours prior to bombardment. It iscontemplated that pretreatment of cells with other osmotically activesolutes for a period of 1-6 hours also may be desirable.

[0260] 2. Plasmid Configuration

[0261] In some instances, it will be desirable to deliver DNA to cellsthat does not contain DNA sequences necessary for maintenance of theplasmid vector in the bacterial host, e.g., E. coli, such as antibioticresistance genes, including but not limited to ampicillin, kanamycin,and tetracycline resistance, and prokaryotic origins of DNA replication.In such case, a DNA fragment containing the transforming DNA may bepurified prior to transformation. An exemplary method of purification isgel electrophoresis on a 1.2% low melting temperature agarose gel,followed by recovery from the agarose gel by melting gel slices in a6-10 fold excess of Tris-EDTA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA,70° C.-72° C.); frozen and thawed (37° C.); and the agarose pelleted bycentrifugation. A Qiagen Q-100 column then may be used for purificationof DNA. For efficient recovery of DNA, the flow rate of the column maybe adjusted to 40 ml/hr.

[0262] Isolated DNA fragments can be recovered from agarose gels using avariety of electroelution techniques, enzyme digestion of the agarose,or binding of DNA to glass beads (e.g., Gene Clean). In addition, HPLCand/or use of magnetic particles may be used to isolate DNA fragments.As an alternative to isolation of DNA fragments, a plasmid vector can bedigested with a restriction enzyme and this DNA delivered to cellswithout prior purification of the expression cassette fragment.

[0263] V. Recipient Cells for Transformation

[0264] Tissue culture requires media and controlled environments.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism. Themedium usually is a suspension of various categories of ingredients(salts, amino acids, growth regulators, sugars, buffers) that arerequired for growth of most cell types. However, each specific cell typerequires a specific range of ingredient proportions for growth, and aneven more specific range of formulas for optimum growth. Rate of cellgrowth also will vary among cultures initiated with the array of mediathat permit growth of that cell type.

[0265] Nutrient media is prepared as a liquid, but this may besolidified by adding the liquid to materials capable of providing asolid support. Agar is most commonly used for this purpose. Bactoagar,Hazelton agar, Gelrite, and Gelgro are specific types of solid supportthat are suitable for growth of plant cells in tissue culture.

[0266] Some cell types will grow and divide either in liquid suspensionor on solid media. As disclosed herein, plant cells will grow insuspension or on solid medium, but regeneration of plants fromsuspension cultures typically requires transfer from liquid to solidmedia at some point in development. The type and extent ofdifferentiation of cells in culture will be affected not only by thetype of media used and by the environment, for example, pH, but also bywhether media is solid or liquid. Table 8 illustrates the composition ofvarious media useful for creation of recipient cells and for plantregeneration.

[0267] Recipient cell targets include, but are not limited to, meristemcells, Type I, Type II, and Type III callus, immature embryos andgametic cells such as microspores, pollen, sperm and egg cells. It iscontemplated that any cell from which a fertile plant may be regeneratedis useful as a recipient cell. Type I, Type II, and Type III callus maybe initiated from tissue sources including, but not limited to, immatureembryos, immature inflorescenses, seedling apical meristems, microsporesand the like. Those cells that are capable of proliferating as callusalso are recipient cells for genetic transformation. The presentinvention provides techniques for transforming immature embryos andsubsequent regeneration of fertile transgenic plants. Transformation ofimmature embryos obviates the need for long term development ofrecipient cell cultures. Pollen, as well as its precursor cells,microspores, may be capable of functioning as recipient cells forgenetic transformation, or as vectors to carry foreign DNA forincorporation during fertilization. Direct pollen transformation wouldobviate the need for cell culture. Meristematic cells (i.e., plant cellscapable of continual cell division and characterized by anundifferentiated cytological appearance, normally found at growingpoints or tissues in plants such as root tips, stem apices, lateralbuds, etc.) may represent another type of recipient plant cell. Becauseof their undifferentiated growth and capacity for organ differentiationand totipotency, a single transformed meristematic cell could berecovered as a whole transformed plant. In fact, it is proposed thatembryogenic suspension cultures may be an in vitro meristematic cellsystem, retaining an ability for continued cell division in anundifferentiated state, controlled by the media environment.

[0268] Cultured plant cells that can serve as recipient cells fortransforming with desired DNA segments may be any plant cells includingmaize cells, and more specifically, cells from Zea mays L. Somatic cellsare of various types. Embryogenic cells are one example of somatic cellsthat may be induced to regenerate a plant through embryo formation.Non-embryogenic cells are those that typically will not respond in sucha fashion. An example of non-embryogenic cells are certain Black MexicanSweet (BMS) corn cells.

[0269] The development of embryogenic calli and suspension culturesuseful in the context of the present invention, e.g., as recipient cellsfor transformation, has been described in U.S. Pat. No. 5,134,074; U.S.Pat. No. 5,489,520 and U.S. Pat. No. 5,990,390; each of which isincorporated herein by reference in its entirety.

[0270] Certain techniques may be used that enrich recipient cells withina cell population. For example, Type II callus development, followed bymanual selection and culture of friable, embryogenic tissue, generallyresults in an enrichment of recipient cells for use in microprojectiletransformation. Suspension culturing, particularly using the mediadisclosed herein, may improve the ratio of recipient to non-recipientcells in any given population. Manual selection techniques that can beemployed to select recipient cells may include, e.g., assessing cellmorphology and differentiation, or may use various physical orbiological means. Cryopreservation also is a possible method ofselecting for recipient cells.

[0271] Manual selection of recipient cells, e.g., by selectingembryogenic cells from the surface of a Type II callus, is one meansthat may be used in an attempt to enrich for recipient cells prior toculturing (whether cultured on solid media or in suspension). Thepreferred cells may be those located at the surface of a cell cluster,and may further be identifiable by their lack of differentiation, theirsize and dense cytoplasm. The preferred cells will generally be thosecells that are less differentiated, or not yet committed todifferentiation. Thus, one may wish to identify and select those cellsthat are cytoplasmically dense, relatively unvacuolated with a highnucleus to cytoplasm ratio (e.g., determined by cytologicalobservations), small in size (e.g., 10-20 μm), and capable of sustaineddivisions and somatic proembryo formation.

[0272] It is proposed that other means for identifying such cells alsomay be employed. For example, through the use of dyes, such as Evan'sblue, which are excluded by cells with relatively non-permeablemembranes, such as embryogenic cells, and taken up by relativelydifferentiated cells such as root-like cells and snake cells (so-calleddue to their snake-like appearance).

[0273] Other possible means of identifying recipient cells include theuse of isozyme markers of embryogenic cells, such as glutamatedehydrogenase, which can be detected by cytochemical stains (Fransz etal., 1989). However, it is cautioned that the use of isozyme markersincluding glutamate dehydrogenase may lead to some degree of falsepositives from non-embryogenic cells such as rooty cells thatnonetheless have a relatively high metabolic activity.

[0274] A. Culturing Cells to be Recipients for Transformation

[0275] The ability to prepare and cryopreserve cultures of plant cellsis important to certain aspects of the present invention, in that itprovides a means for reproducibly and successfully preparing cells fortransformation. A variety of different types of media have beenpreviously developed and may be employed in carrying out various aspectsof the invention. The following table, Table 8, sets forth thecomposition of the media preferred by the inventor for carrying outthese aspects of the invention. TABLE 8 Tissue Culture Media Used forType II Callus Development, Development of Suspension Cultures andRegeneration of Plant Cells (Particularly Cells) OTHER BASALCOMPONENTS** MEDIA NO. MEDIUM SUCROSE pH (Amount/L) 7  MS* 2% 6.0 .25 mgthiamine .5 mg BAP .5 mg NAA Bactoagar 10 MS 2% 6.0 .25 mg thiamine 1 mgBAP 1 mg 2,4-D 400 mg L-proline Bactoagar 19 MS 2% 6.0 .25 mg thiamine.25 mg BAP .25 mg NAA Bactoagar 20 MS 3% 6.0 .25 mg thiamine 1 mg BAP 1mg NAA Bactoagar 52 MS 2% 6.0 .25 mg thiamine 1 mg 2,4-D 10⁻⁷ M ABABACTOAGAR 101 MS 3% 6.0 MS vitamins 100 mg myo-inositol Bactoagar 105 MS— 3.5 0.04 mg NAA 3 mg BAP 1 mg thiamine.HCl 0.5 mg niacin 0.91 mgL-asparagine monohydrate 100 mg myo-inositol 100 mg casein hydrolysate1.4 g L-proline 20 g sorbitol 2.0 g Gelgro 110 MS 6% 5.8 1 mgthiamine.HCl 1 mg niacin 3.6 g Gelgro 142 MS 6% 6.0 MS vitamins 5 mg BAP0.186 mg NAA 0.175 mg IAA 0.403 mg 2IP Bactoagar 157 MS 6% 6.0 MSvitamins 100 mg myo-inositol Bactoagar 163 MS 3% 6.0 MS vitamins 3.3 mgdicamba 100 mg myo-inositol Bactoagar 171 MS 3% 6.0 MS vitamins .25 mg2,4-D 10 mg BAP 100 mg myo-inositol Bactoagar 173 MS 6% 6.0 MS vitamins5 mg BAP .186 mg NAA .175 mg IAA .403 mg 2IP 10⁻⁷ M ABA 200 mgmyo-inositol Bactoagar 177 MS 3% 6.0 MS vitamins .25 mg 2,4-D 10 mg BAP10⁻⁷ M ABA 100 mg myo-inositol Bactoagar 185 MS — 5.8 3 mg BAP .04 mgNAA RT vitamins 1.65 mg thiamine 1.38 g L-proline 20 g sorbitolBactoagar 189 MS — 5.8 3 mg BAP .04 mg NAA .5 mg niacin 800 mgL-asparagine 100 mg casamino acids 20 g sorbitol 1.4 g L-proline 100 mgmyo-inositol Gelgro 201 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D100 mg casein hydrolysate 2.9 g L-proline Gelgro 205 N6 2% 5.8 N6vitamins 2 mg L-glycine .5 mg 2,4-D 100 mg casein hydrolysate 2.9 gL-proline Gelgro 209 N6 6% 5.8 N6 vitamins 2 mg L-glycine 100 mg caseinhydrolysate 0.69 g L-proline Bactoagar 210 N6 3% 5.5 N6 vitamins 2 mg2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 790 mg L-asparagine 100mg casein hydrolysate 1.4 g L-proline Hazelton agar**** 2 mg L-glycine211 N6 2% 5.8 1 mg 2,4-D 0.5 mg niacin 1.0 mg thiamine 0.91 gL-asparagine 100 mg myo-inositol 0.5 g MES 100 mg/L casein hydrolysate1.6 g MgCl₂—6H₂O 0.69 g L-proline 2 g Gelgro 212 N6 3% 5.5 N6 vitamins 2mg L-glycine 2 mg 2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 100mg casein hydrolysate 1.4 g L-proline Hazelton agar**** 227 N6 2% 5.8 N6vitamins 2 mg L-glycine 13.2 mg dicamba 100 mg casein hydrolysate 2.9 gL-proline Gelgro 273 (also, N6 2% 5.8 N6 vitamins 201V, 236S, 2 mgL-glycine 201D, 2071, 1 mg 2,4-D 2366, 201SV, 16.9 mg AgNO₃ 2377, and100 mg casein 201BV) hydrolysate 2.9 g L-proline 279 N6 2% 5.8 3.3 mgdicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100 mg caseinhydrolysate 100 mg myoinositol 1.4 g L-proline Gelgro**** 288 N6 3% 3.3mg dicamba 1 mg thiamine .5 mg niacin .8 g L-asparagine 100 mgmyo-inosital 1.4 g L-proline 100 mg casein hydrolysate 16.9 mg AgNO₃Gelgro 401 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D 2 mg NAA200 mg casein hydrolysate 500 mg K₂SO₄ 400 mg KH₂PO₄ 100 mg myo-inositol402 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D 200 mg caseinhydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mg KH₂PO₄ 100 mgmyo-inositol 409 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 9.9 mgdicamba 200 mg casein hydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mgKH₂PO₄ 100 mg myo-inositol 501 Clark's 2% 5.7 Medium** 607 ½ × MS 3% 5.81 mg thiamine 1 mg niacin Gelrite 615 MS 3% 6.0 MS vitamins 6 mg BAP 100mg myo-mositol Bactoagar 617 ½ × MS   1.5% 6.0 MS vitamins 50 mgmyo-inositol Bactoagar 708 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg2,4-D 200 mg casein hydrolysate 0.69 g L-proline Gelrite 721 N6 2% 5.83.3 mg dicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100 mgmyo-inositol 100 mg casein hydrolysate 1.4 g L-proline 54.65 g mannitolGelgro 726 N6 3% 5.8 3.3 mg dicamba .5 mg niacin 1 mg thiamine 800 mgL-asparagine 100 mg myo-inositol 100 mg casein hydrolysate 1.4 gL-proline 727 N6 3% 5.8 N6 vitamins 2 mg L-glycine 9.9 mg dicamba 100 mgcasein hydrolysate 2.9 g L-proline Gelgro 728 N6 3% 5.8 N6 vitamins 2 mgL-glycine 9.9 mg dicamba 16.9 mg AgNO₃ 100 mg casein hydrolysate 2.9 gL-proline Gelgro

[0276] OTHER BASAL COMPONENTS** MEDIA NO. MEDIUM SUCROSE pH (Amount/L)734 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg 2,4-D 14 g Fesequestreene (replaces Fe-EDTA) 200 mg casein hydrolyste 0.69 gL-proline Gelrite 735 N6 2% 5.8 1 mg 2,4-D .5 mg niacin .91 gL-asparagine 100 mg myo-inositol 1 mg thiamine .5 g MES .75 g MgCl₂ 100mg casein hydrolysate 0.69 g L-proline Gelgro 2004 N6 3% 5.8 1 mgthiamine 0.5 mg macin 3.3 mg dicamba 17 mg AgNO₃ 1.4 g L-proline 0.8 gL-asparagine 100 mg casein hydrolysate 100 mg myo-inositol Gelrite 2008N6 3% 5.8 1 mg thiamine 0.5 mg niacin 3.3 mg dicamba 1.4 g L-proline 0.8g L-asparagine Gelrite # myo-inositol and glycine.

[0277] A number of exemplary cultures that may be used fortransformation have been developed and are disclosed in U.S. Pat. No.5,590,390, the disclosure of which is specifically incorporated hereinby reference.

[0278] B. Media

[0279] In certain embodiments of the current invention, recipient cellsmay be selected following growth in culture. Where employed, culturedcells may be grown either on solid supports or in the form of liquidsuspensions. In either instance, nutrients may be provided to the cellsin the form of media, and environmental conditions controlled. There aremany types of tissue culture media comprised of various amino acids,salts, sugars, growth regulators and vitamins. Most of the mediaemployed in the practice of the invention will have some similarcomponents (see Table 8), but may differ in the composition andproportions of their ingredients depending on the particular applicationenvisioned. For example, various cell types usually grow in more thanone type of media, but will exhibit different growth rates and differentmorphologies, depending on the growth media. In some media, cellssurvive but do not divide.

[0280] Various types of media suitable for culture of plant cellspreviously have been described. Examples of these media include, but arenot limited to, the N6 medium described by Chu et al. (1975) and MSmedia (Murashige and Skoog, 1962). It has been discovered that mediasuch as MS that have a high ammonia/nitrate ratio are counterproductiveto the generation of recipient cells in that they promote loss ofmorphogenic capacity. N6 media, on the other hand, has a somewhat lowerammonia/nitrate ratio, and is contemplated to promote the generation ofrecipient cells by maintaining cells in a proembryonic state capable ofsustained divisions.

[0281] C. Maintenance

[0282] The method of maintenance of cell cultures may contribute totheir utility as sources of recipient cells for transformation. Manualselection of cells for transfer to fresh culture medium, frequency oftransfer to fresh culture medium, composition of culture medium, andenvironmental factors including, but not limited to, light quality andquantity and temperature are all important factors in maintaining callusand/or suspension cultures that are useful as sources of recipientcells. It is contemplated that alternating callus between differentculture conditions may be beneficial in enriching for recipient cellswithin a culture. For example, it is proposed that cells may be culturedin suspension culture, but transferred to solid medium at regularintervals. After a period of growth on solid medium cells can bemanually selected for return to liquid culture medium. It is proposedthat by repeating this sequence of transfers to fresh culture medium itis possible to enrich for recipient cells. It also is contemplated thatpassing cell cultures through a 1.9 mm sieve is useful in maintainingthe friability of a callus or suspension culture and may be beneficialin enriching for transformable cells.

[0283] D. Cryopreservation Methods

[0284] Cryopreservation is important because it allows one to maintainand preserve a known transformable cell culture for future use, whileeliminating the cumulative detrimental effects associated with extendedculture periods.

[0285] Cell suspensions and callus were cryopreserved usingmodifications of methods previously reported (Finkle, 1985; Withers &King, 1979). The cryopreservation protocol comprised adding a pre-cooled(0° C.) concentrated cryoprotectant mixture stepwise over a period ofone to two hours to pre-cooled (0° C.) cells. The mixture was maintainedat 0° C. throughout this period. The volume of added cryoprotectant wasequal to the initial volume of the cell suspension (1: 1 addition), andthe final concentration of cryoprotectant additives was 10% dimethylsulfoxide, 10% polyethylene glycol (6000 MW), 0.23 M proline and 0.23 Mglucose. The mixture was allowed to equilibrate at 0° C. for 30 minutes,during which time the cell suspension/cryoprotectant mixture was dividedinto 1.5 ml aliquot (0.5 ml packed cell volume) in 2 ml polyethylenecryo-vials. The tubes were cooled at 0.5° C./minute to −8° C. and heldat this temperature for ice nucleation.

[0286] Once extracellular ice formation had been visually confirmed, thetubes were cooled at 0.5° C./minute from −8° C. to −35° C. They wereheld at this temperature for 45 minutes (to insure uniformfreeze-induced dehydration throughout the cell clusters). At this point,the cells had lost the majority of their osmotic volume (i.e., there islittle free water left in the cells), and they could be safely plungedinto liquid nitrogen for storage. The paucity of free water remaining inthe cells in conjunction with the rapid cooling rates from −35° C. to−196° C. prevented large organized ice crystals from forming in thecells. The cells are stored in liquid nitrogen, which effectivelyimmobilizes the cells and slows metabolic processes to the point wherelong-term storage should not be detrimental.

[0287] Thawing of the extracellular solution was accomplished byremoving the cryo-tube from liquid nitrogen and swirling it in sterile42° C. water for approximately 2 minutes. The tube was removed from theheat immediately after the last ice crystals had melted to preventheating the tissue. The cell suspension (still in the cryoprotectantmixture) was pipetted onto a filter, resting on a layer of BMS cells(the feeder layer that provided a nurse effect during recovery). Thecryoprotectant solution is removed by pipetting. Culture mediumcomprised a callus proliferation medium with increased osmotic strength.Dilution of the cryoprotectant occurred slowly as the solutes diffusedaway through the filter and nutrients diffused upward to the recoveringcells. Once subsequent growth of the thawed cells was noted, the growingtissue was transferred to fresh culture medium. If initiation of asuspension culture was desired, the cell clusters were transferred backinto liquid suspension medium as soon as sufficient cell mass had beenregained (usually within 1 to 2 weeks). Alternatively, cells werecultured on solid callus proliferation medium. After the culture wasreestablished in liquid (within 1 to 2 additional weeks), it was usedfor transformation experiments. When desired, previously cryopreservedcultures may be frozen again for storage.

[0288] VI. Production and Characterization of Stably Transformed Plants

[0289] After effecting delivery of exogenous DNA to recipient cells, thenext steps generally concern identifying the transformed cells forfurther culturing and plant regeneration. As mentioned herein, in orderto improve the ability to identify transformants, one may desire toemploy a selectable or screenable marker gene as, or in addition to, theexpressible gene of interest. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

[0290] A. Selection

[0291] It is believed that DNA is introduced into only a smallpercentage of target cells in any one experiment. In order to provide anefficient system for identification of those cells receiving DNA andintegrating it into their genomes one may employ a means for selectingthose cells that are stably transformed. One exemplary embodiment ofsuch a method is to introduce into the host cell, a marker gene thatconfers resistance to some normally inhibitory agent, such as anantibiotic or herbicide. Examples of antibiotics that may be usedinclude the aminoglycoside antibiotics neomycin, kanamycin andparomomycin, or the antibiotic hygromycin. Resistance to theaminoglycoside antibiotics is conferred by aminoglycosidephosphostransferase enzymes such as neomycin phosphotransferase II (NPTII) or NPT I, whereas resistance to hygromycin is conferred byhygromycin phosphotransferase.

[0292] Potentially transformed cells then are exposed to the selectiveagent. In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA. Usingthe techniques disclosed herein, greater than 40% of bombarded embryosmay yield transformants.

[0293] One herbicide that constitutes a desirable selection agent is thebroad spectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide LIBERTY® also is effectiveas a selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

[0294] The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) that is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in U.S. Pat. No. 5,276,268, wherein thegene is isolated from Streptomyces viridochromogenes. In the bacterialsource organism, this enzyme acetylates the free amino group of PPTpreventing auto-toxicity (Thompson et al., 1987). The bar gene has beencloned (Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants that expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

[0295] Another example of a herbicide that is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS, which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations that confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, PCT PublicationNo. WO 97/04103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT Publication No. WO97/04103). Furthermore, a naturally occurringglyphosate resistant EPSPS may be used, e.g., the CP4 gene isolated fromAgrobacterium encodes a glyphosate resistant EPSPS (U.S. Pat. No.5,627,061).

[0296] To use the nptII-paromomycin selective system, bombarded tissueis cultured for 0-28 days, preferably 0-10 days, most preferably lessthan 1 day on culture medium lacking paromomycin. Bombarded tissue istransferred to culture medium comprising 25-500 mg/L paromomycin andsubculture at 1 to 3 week intervals onto fresh selective medium for 3-15weeks. Transformants are visually identified as healthy growing callus.

[0297] To use the bar-bialaphos or the EPSPS-glyphosate selectivesystem, bombarded tissue is cultured for 0-28 days on nonselectivemedium and subsequently transferred to medium containing from 1-3 mg/lbialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/lbialaphos or 1-3 mM glyphosate will typically be preferred, it isproposed that ranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosatewill find utility in the practice of the invention. Tissue can be placedon any porous, inert, solid or semi-solid support for bombardment,including but not limited to filters and solid culture medium. Bialaphosand glyphosate are provided as examples of agents suitable for selectionof transformants, but the technique of this invention is not limited tothem.

[0298] Although general methods of use of nptII, bar and EPSPS asselectable marker genes are described above, following DNA delivery bymicroprojectile bombment it is recognized that the described selectionmethods will work following DNA delivery by any method, including butnot limited to, microprojectile bombardment, Agrobacterium mediatedtransformation, and other methods of DNA delivery to plant cells areknown in the art.

[0299] It further is contemplated that the herbicide dalapon,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (Buchanan-Wollastonet al., 1992; U.S. Pat. No. 5,780,708).

[0300] Alternatively, a gene encoding anthranilate synthase, whichconfers resistance to certain amino acid analogs, e.g.,5-methyltryptophan or 6-methyl anthranilate, may be useful as aselectable marker gene. The use of an anthranilate synthase gene as aselectable marker was described in U.S. Pat. No. 5,508,468 and PCTPublication No. WO 97/26366.

[0301] An example of a screenable marker trait is the red pigmentproduced under the control of the R-locus in maize. This pigment may bedetected by culturing cells on a solid support containing nutrient mediacapable of supporting growth at this stage and selecting cells fromcolonies (visible aggregates of cells) that are pigmented. These cellsmay be cultured further, either in suspension or on solid media. TheR-locus is useful for selection of transformants from bombarded immatureembryos. In a similar fashion, the introduction of the C1 and B geneswill result in pigmented cells and/or tissues.

[0302] The enzyme luciferase may be used as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light that can be detectedon photographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellsthat are expressing luciferase and manipulate those in real time.Another screenable marker that may be used in a similar fashion is thegene coding for green fluorescent protein.

[0303] It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and non-transforrnants alike, thus causingthe selection technique to not be effective. It is proposed thatselection with a growth inhibiting compound, such as bialaphos orglyphosate at concentrations below those that cause 100% inhibitionfollowed by screening of growing tissue for expression of a screenablemarker gene such as luciferase would allow one to recover transformantsfrom cell or tissue types that are not amenable to selection alone. Itis proposed that combinations of selection and screening may enable oneto identify transformants in a wider variety of cell and tissue types.This may be efficiently achieved using a gene fusion between aselectable marker gene and a screenable marker gene, for example,between an NPTII gene and a GFP gene.

[0304] B. Regeneration and Seed Production

[0305] Cells that survive the exposure to the selective agent, or cellsthat have been scored positive in a screening assay, may be cultured inmedia that supports regeneration of plants. In an exemplary embodiment,MS and N6 media may be modified (see Table 8) by including furthersubstances such as growth regulators. A preferred growth regulator forsuch purposes is dicamba or 2,4-D. However, other growth regulators maybe employed, including NAA, NAA+2,4-D or perhaps even picloram. Mediaimprovement in these and like ways has been found to facilitate thegrowth of cells at specific developmental stages. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least 2 wk, then transferred to mediaconducive to maturation of embryoids. Cultures are transferred every 2wk on this medium. Shoot development will signal the time to transfer tomedium lacking growth regulators.

[0306] The transformed cells, identified by selection or screening andcultured in an appropriate medium that supports regeneration, will thenbe allowed to mature into plants. Developing plantlets are transferredto soiless plant growth mix, and hardened off, e.g., in anenvironmentally controlled chamber at about 85% relative humidity, 600ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transferto a greenhouse or growth chamber for maturation. Plants are preferablymatured either in a growth chamber or greenhouse. Plants are regeneratedfrom about 6 wk to 10 months after a transformant is identified,depending on the initial tissue. During regeneration, cells are grown onsolid media in tissue culture vessels. Illustrative embodiments of suchvessels are petri dishes and Plant Cons. Regenerating plants arepreferably grown at about 19 to 28° C. After the regenerating plantshave reached the stage of shoot and root development, they may betransferred to a greenhouse for further growth and testing.

[0307] Note, however, that seeds on transformed plants may occasionallyrequire embryo rescue due to cessation of seed development and prematuresenescence of plants. To rescue developing embryos, they are excisedfrom surface-disinfected seeds 10-20 days post-pollination and cultured.An embodiment of media used for culture at this stage comprises MSsalts, 2% sucrose, and 5.5 g/l agarose. In embryo rescue, large embryos(defined as greater than 3 mm in length) are germinated directly on anappropriate media. Embryos smaller than that may be cultured for 1 wk onmedia containing the above ingredients along with 10⁻⁵M abscisic acidand then transferred to growth regulator-free medium for germination.

[0308] Progeny may be recovered from transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R_(O)) and their progeny of any generation tested exhibited nobialaphos-related necrosis after localized application of the herbicideBasta to leaves, if there was functional PAT activity in the plants asassessed by an in vitro enzymatic assay. All PAT positive progeny testedcontained bar, confirming that the presence of the enzyme and theresistance to bialaphos were associated with the transmission throughthe germline of the marker gene.

[0309] C. Characterization

[0310] To confirm the presence of the isolated and purified DNAsegment(s) or “transgene(s)” in the regenerating plants, a variety ofassays may be performed. Such assays include, for example, “molecularbiological” assays well known to those of skill in the art, such asSouthern and Northern blotting, RT-PCR and PCR; “biochemical” assays,such as detecting the presence of a protein product, e.g., byimmunological means (ELISAs and Western blots) or by enzymatic function;plant part assays, such as leaf or root assays; and also, by analyzingthe phenotype of the whole regenerated plant.

[0311] Whereas DNA analysis techniques may be conducted using DNAisolated from any part of a plant, RNA may only be expressed inparticular cells or tissue types and hence it will be necessary toprepare RNA for analysis from these tissues. PCR techniques may also beused for detection and quantitation of RNA produced from introducedisolated and purified DNA segments. In this application of PCR it isfirst necessary to reverse transcribe RNA into DNA, using enzymes suchas reverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique will demonstrate the presence of anRNA species and give information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and will only demonstrate the presence or absenceof an RNA species.

[0312] While Southern blotting and PCR may be used to detect theisolated and purified DNA segment in question, they do not provideinformation as to whether the isolated and purified DNA segment is beingexpressed. Expression may be evaluated by specifically identifying theprotein products of the introduced isolated and purified DNA sequencesor evaluating the phenotypic changes brought about by their expression.

[0313] Assays for the production and identification of specific proteinsmay make use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

[0314] Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of isolated andpurified DNA segments encoding storage proteins that change amino acidcomposition and may be detected by amino acid analysis.

[0315] 1. DNA Integration, RNA Expression and Inheritance

[0316] Genomic DNA may be isolated from callus cell lines or any plantparts to determine the presence of the isolated and purified DNA segmentthrough the use of techniques well known to those skilled in the art.Note that intact sequences will not always be present, presumably due torearrangement or deletion of sequences in the cell.

[0317] The presence of DNA elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of DNA are amplified anddetected by gel electrophoresis or other methods known to the art. Thistype of analysis permits one to determine whether an isolated andpurified DNA segment is present in a stable transformant, but does notprove integration of the introduced isolated and purified DNA segmentinto the host cell genome. It is contemplated that using PCR techniquesit would be possible to clone fragments of the host genomic DNA adjacentto an introduced isolated and purified DNA segment.

[0318] Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced isolated andpurified DNA segments in high molecular weight DNA, i.e., confirm thatthe introduced isolated and purified DNA segment has been integratedinto the host cell genome. The technique of Southern hybridizationprovides information that is obtained using PCR, e.g., the presence ofan isolated and purified DNA segment, but also demonstrates integrationinto the genome and characterizes each individual transformant.

[0319] It is contemplated that using the techniques of dot or slot blothybridization that are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of an isolated and purified DNA segment.However, it is well known in the art that dot or slot blot hybridizationmay produce misleading results, as probe may be non-specifically boundby high molecular weight DNA.

[0320] Both PCR and Southern hybridization techniques can be used todemonstrate transmission of an isolated and purified DNA segment toprogeny. In most instances the characteristic Southern hybridizationpattern for a given transformant will segregate in progeny as one ormore Mendelian genes (Spencer et al., 1992; Laursen et al., 1994)indicating stable inheritance of the gene. For example, in one study, of28 progeny (R₁) plants tested, 50% (N=14) contained bar, confirmingtransmission through the germline of the marker gene. The nonchimericnature of the callus and the parental transforrnants (R₀) was suggestedby germline transmission and the identical Southern blot hybridizationpatterns and intensities of the transforming DNA in callus, R₀ plantsand R₁ progeny that segregated for the transformed gene.

[0321] Whereas DNA analysis techniques may be conducted using DNAisolated from any part of a plant, RNA may only be expressed inparticular cells or tissue types and hence it will be necessary toprepare RNA for analysis from these tissues. PCR techniques may also beused for detection and quantitation of RNA produced from introducedisolated and purified DNA segments. In this application of PCR it isfirst necessary to reverse transcribe RNA into DNA, using enzymes suchas reverse transcriptase, and then, through the use of conventional PCRtechniques, on amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique will demonstrate the presence of anRNA species and give information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and will only demonstrate the presence or absenceof an RNA species.

[0322] 2. Gene Expression

[0323] While Southern blotting and PCR may be used to detect theisolated and purified DNA segment in question, they do not provideinformation as to whether the isolated and purified DNA segment is beingexpressed. Expression may be evaluated by specifically identifying theprotein products of the introduced isolated and purified DNA segments orevaluating the phenotypic changes brought about by their expression.

[0324] Assays for the production and identification of specific proteinsmay make use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

[0325] Assay procedures may also be used to identify the expression ofproteins by their functionality, especially the ability of enzymes tocatalyze specific chemical reactions involving specific substrates andproducts. These reactions may be followed by providing and quantifyingthe loss of substrates or the generation of products of the reactions byphysical or chemical procedures. Examples are as varied as the enzyme tobe analyzed and may include assays for PAT enzymatic activity byfollowing production of radio labeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

[0326] Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of isolated andpurified DNA segments encoding enzymes or storage proteins which changeamino acid composition and may be detected by amino acid analysis, or byenzymes that change starch quantity that may be analyzed by nearinfrared reflectance spectrometry. Morphological changes may includegreater stature or thicker stalks. Most often changes in response ofplants or plant parts to imposed treatments are evaluated undercarefully controlled conditions termed bioassays.

[0327] D. Establishment of the Introduced DNA in Other Plant Varieties

[0328] Fertile, transgenic plants may then be used in a conventionalbreeding program in order to incorporate the isolated and purified DNAsegment into the desired lines or varieties. Methods and references forconvergent improvement of are given by Hallauer et al. (1988),incorporated herein by reference. Among the approaches that conventionalbreeding programs employ is a conversion process (backcrossing).Briefly, conversion is performed by crossing the initial transgenicfertile plant to elite inbred lines (which may or may not be transgenic)to yield an F₁ hybrid plant. The progeny from this cross will segregatesuch that some of the plants will carry the isolated and purified DNAsegment whereas some will not. The plants that do carry the isolated andpurified DNA segment are then crossed again to the elite inbred linesresulting in progeny that segregate once more. This backcrossing processis repeated until the original elite inbred has been converted to a linecontaining the isolated and purified DNA segment, yet possessing allimportant attributes originally found in the parent. Generally, thiswill require about 6-8 generations. Then the resultant F_(n) hybrid isusually selfed 5-7 times to yield an inbred line. A separatebackcrossing program will be generally used for every elite line that isto be converted to a genetically engineered elite line.

[0329] Generally, the commercial value of the transformed plantsproduced herein will be greatest if the isolated and purified DNAsegment can be incorporated into many different hybrid combinations. Afarmer typically grows several hybrids based on differences in maturity,standability, and other agronomic traits. Also, the farmer must select ahybrid based upon his or her geographic location since hybrids adaptedto one region are generally not adapted to another because ofdifferences in such traits as maturity, disease, drought and insectresistance. As such, it is necessary to incorporate the gene into alarge number of parental lines so that many hybrid combinations can beproduced containing the isolated and purified DNA segment.

[0330] Plant breeding and the techniques and skills required to transfergenes from one line or variety to another are well known to thoseskilled in the art. Thus, introducing an isolated and purified DNAsegment, preferably in the form of recombinant DNA, into any other lineor variety can be accomplished by these breeding procedures.

[0331] E. Alteration of Transgene Insertions

[0332] At anytime during the process of incorporation of a transgeneinto other varieties of the plant species, alterations in the transgeneinsertion may be identified or selected. Preferably, alterations areinduced early in the process of incorporating the transgene insertioninto other varieties, so as to minimize the number of further varietyconversions, e.g., backcross conversions, that must be made after thealtered transgene insertion is selected. The use of homologousrecombination to alter a transgene insertion requires the presence of adirectly repeated DNA sequence within the transgene insertion. Directlyrepeated sequences may be present on a plasmid vector when introduced ina plant. For example, plasmid pMON19344 (FIG. 4) comprises a cryIA(b)gene flanked by directly repeated P-e35S and hsp70 intron sequences.Furthermore, the nptII gene in pMON19344 is flanked by directly repeatednopaline synthase (NOS) 3′ sequences. Similarly, a single transgene on aplasmid vector may be flanked by directly repeated sequences.Integration of plasmid vectors such as pMON19344 as a linear transgeneinsertion leads to both the cryIA(b) and nptII genes being flanked bydirectly repeated sequences.

[0333] Alternatively, directly repeated DNA sequences may be generatedin the transgene insertion process by rearrangements, duplications andtandem integrations of DNA sequences in the transgene insertion.Therefore, although directly repeated DNA sequences are not present onthe plasmid vectors that are introduced into the plants, direct repeatsare produced during the DNA integration process. Regardless of theprocess used, the result is a transgene insertion comprising directlyrepeated DNA sequences.

[0334] In order for recombination between direct repeats to delete atransgene and be passed on to the next generation, the recombinationevent must occur in the germline, or upstream of the germline and thenenter the germline. Although somatic recombination is known in plants(Evans and Paddock, 1979; Peterhans et al., 1990; Gal et el., 1991;Assad and Signer, 1992; Swoboda et al, 1993; Swoboda et al., 1994;Jelesko et al., 1999; Zubko et al., 2000), it is likely thatrecombination frequencies are elevated in meiosis. However, it isadvantageous to screen and select for altered transgene insertionsamongst a population of transgenic events which has been previouslydetermined to express a transgene at a level to confer a desiredphenotype. Screening and selection for transgene insertion eventalteration is, therefore, preferably done in plants and most preferablydone in plants for which transgene expression data is known. Sinceseveral recombinational pathways can lead to deletion (FIGS. 1-3, Table2) it is not necessary for the transgene to be homozygous, althoughpassing through a homozygous stage may facilitate screening for loss ofa transgene when homozygous plants are outcrossed.

[0335] In the normal course of plant transformation a transgeneinsertion occurs at a single chromosomal locus. Therefore, thetransformed cell and directly derived transformed plant contain a singlecopy of the transgene insertion, i.e., the cell and plant arehemizygous. Plants in which recombination between direct repeats havedeleted a transgene may be identified in progeny produced throughself-fertilization or outcrossing to a plant lacking the transgeneinsertion. Preferably, plants comprising homozygous transgenicinsertions are crossed to non-transgenic plants in order to simplifyidentification of recombinants.

[0336] During the process of meiotic recombination, many types ofrecombination are possible, including equal recombination betweenchromosome alleles, also known as allelic recombination. It isanticipated that allelic recombinants will demonstrate gene expressionsimilar to the parent plant. Selection of progeny plants comprisingaltered transgene insertions produced through recombination betweendirect repeats, resulting in the loss of a transgene, is based onidentification of progeny plants with altered transgene expression,preferably loss of transgene expression. Altered expression may bedetected by a phenotypic assay, e.g., herbicide resistance or insectresistance, or direct assays for enzyme activity or presence of thetransgene encoded protein. The presence of an altered transgeneinsertion is likely in progeny plants in which transgene expressiondiffers from expression in the parent transgenic plant. Alterations inthe transgene insertion may be confirmed by PCR or Southern blotanalysis.

[0337] Alteration of transgene insertion event structure may also beobserved in cultured cells, such as callus, following homologousrecombination between directly repeated DNA sequences. Therefore, onlyplants with the desired transgene insertion structure are regenerated.Furthermore, because the insertion alteration occurs in vitro, it is notnecessary to segregate unlinked undesirable loci, thereby acceleratingthe process of generating altered transgene insertion events.

[0338] It is possible to enhance the frequency of homologousrecombination between directly repeated DNA sequences. For example,expression of the E. coli recA or ruvC genes in plants has beendemonstrated to increase ten-fold the frequency of homologousrecombination between directly repeated DNA sequences (Reiss et al.,1996; Shalev et al., 1999).

[0339] If alteration of transgene insertion events occurs in culturedcells, it is desirable to select for the product of the direct repeatrecombination, e.g., deletion of a DNA sequence within the transgeneinsertion. A preferred method of selecting for a transgene deletionderivative is to include a negative selectable marker gene within theDNA sequence to be deleted. In the presence of a negative selectionagent, cells expressing the negative selectable marker gene are killedand, therefore, in the absence of gene expression cells survive. Forexample, the compound glyceryl glyphosate is not toxic to plant cells.However, the Burkholderia caryophilli PG2982 pehA gene encodes aphosphonate ester hydrolase enzyme that catalyzes the hydrolysis ofglyceryl glyphosate to the toxic compound glyphosate (U.S. Pat. No.5,254,801; Dotson et al., 1996a; Dotson et al. 1996b). Therefore,expression of the pehA gene leads to cell death in the presence ofglyceryl glyphosate, but not in the absence of the compound. Othernegative selectable markers are known to function in plants. Forexample, the enzyme cytosine deaminase converts non-toxic5-fluorocytosine to the toxic compound 5-fluorouracil and has been usedas a negative selectable marker in plants (Stouggard,. 1993). Inaddition, T-DNA gene 2 is useful as a selectable marker in plants(Depicker et al., 1988). The T-DNA gene 2 protein catalyzes theconversion of alpha-napthalene acetamide (NAM) on auxin alpha-napthaleneacetic acid (NAA). NAM is not toxic to plant cells, except in thepresence of T-DNA gene 2 product and high concentrations of NAM, e.g.,30-300 μM. Furthermore, the herpes simplex thymidine kinase gene hasbeen used as a negative selectable marker in plants (Czako and Marton,1994).

[0340] A DNA sequence comprising a positive selectable marker gene,e.g., nptII or another positive selectable marker gene, and a negativeselectable marker gene, e.g., pehA, T-DNA gene 2, cytosine deaminasegene, flanked by a directly repeated DNA sequence is introduced into aplant. Alternatively, a fusion of the positive and negative selectablemarker genes is used. Transformants are identified using an appropriateselective agent-selectable marker gene combination, e.g., kanamycin orparomomycin and the nptII gene. Following identification of transformedcell lines, selection for the negative selectable marker, and thereforedeletion of the negative selectable marker gene, is initiated.Resistance to the negative selection agent is indicative of loss of thenegative selectable marker gene, i.e., transgene deletion. Transgenedeleted cells are also sensitive to the positive selectable marker asboth positive and negative selectable markers present between directlyrepeated DNA sequences are deleted. Selection of transgene deletionderivative events may require removal of the positive selective agentfor a period of time prior to imposing negative selection in order toallow for the occurrence of a transgene deletion recombination event.Alternatively, a gradual decrease in positive selection with aconcomittant increase in negative selection may be used, or the increaseand decrease in positive and negative selection agents may occursimultaneously. FIG. 13 illustrates use of a negative selectable markerto select for cells with altered transgene insertions. Transgene deletedplants are regenerated from cultured cells that are identified followingpositive and negative selection. Selection of transgene deletions usinga negative selectable marker gene was disclosed by Zubko et al., (2000).

[0341] F. Uses of Transgenic Plants

[0342] The transgenic plants produced herein are expected to be usefulfor a variety of commercial and research purposes. Transgenic plants canbe created for use in traditional agriculture to possess traitsbeneficial to the grower (e.g., agronomic traits such as resistance towater deficit, pest resistance, herbicide resistance or increasedyield), beneficial to the consumer of the grain harvested from the plant(e.g., improved nutritive content in human food or animal feed), orbeneficial to the food processor (e.g., improved processing traits). Insuch uses, the plants are generally grown for the use of their grain inhuman or animal foods. However, other parts of the plants, includingstalks, husks, vegetative parts, and the like, may also have utility,including use as part of animal silage or for ornamental purposes.Often, chemical constituents (e.g., oils or starches) of maize and othercrops are extracted for foods or industrial use and transgenic plantsmay be created that have enhanced or modified levels of such components.

[0343] Transgenic plants may also find use in the commercial manufactureof proteins or other molecules, where the molecule of interest isextracted or purified from plant parts, seeds, and the like. Cells ortissue from the plants may also be cultured, grown in vitro, orfermented to manufacture such molecules.

[0344] The transgenic plants may also be used in commercial breedingprograms, or may be crossed or bred to plants of related crop species.Improvements encoded by the isolated and purified DNA segment may betransferred, e.g., from cells of one species to cells of other species,e.g., by protoplast fusion.

[0345] The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences that can be used to identify proprietary lines orvarieties.

[0346] The following examples are illustrative of the present invention.

EXAMPLE 1 Deletion of the Bar Gene from the Transgenic Event DBT418

[0347] Homologous recombination-mediated transgene deletion is a processwhereby the structure of a transgene insert can be altered (see FIG. 1).The process is dependent on the presence of direct repeats of DNAsequences in the transgene insertion. Direct repeats may be present inthe transgene used for transformation, or they may arise throughmulti-element integration at the site of transgene insertion. The directrepeats might be, for example, incomplete parts of a transgene that,upon recombination, produce a complete transgene conferring anidentifiable phenotype.

[0348] Line DBT418 was produced by microprojectile bombardment ofembryogenic cells with plasmid vectors pDPG354 (FIG. 6), pDPG165 (FIG.7) and pDPG320 (FIG. 8). The structure of the transgene insert in theline DBT418 is diagramed in FIG. 9 and described in detail in U.S.D.A.Petition 9629101p for deregulation. The insert has one functional copyof a bar gene conferring resistance to the herbicide phosphinothricin.Flanking the bar gene on both sides are directly repeated DNA sequencesthat include cloning vector DNA and Bt toxin encoding DNA sequences. Inaddition to these direct repeats, there are additional shorter regionsof direct homology that also may serve as target sequences fornon-reciprocal recombination mediated deletion of transgene DNA withinthe insert.

[0349] In order to identify individuals that have undergone homologousrecombination mediated transgene deletion, an assay was carried out toscreen for plants that showed a loss of the phosphinothricin resistancephenotype. Southern blot analysis was used to characterize the copynumber of transgene elements present in the phosphinothricin sensitiveindividuals.

[0350] Hemizygous DBT418 plants were selfed, and progeny identified thatwere homozygous for the DBT418 insertion event. These homozygous plantswere outcrossed to non-transgenic plants to generate a population ofhemizygous seed. Approximately 1,000 seed of a finished inbred,hemizygous for the DBT418 insert, were planted and assayed forphosphinothricin resistance using a nondestructive herbicide leafpainting assay (U.S. Pat. No. 5,489,520). Individuals displaying anecrotic response in the treated area were assayed again by the leafpainting assay for confirmation of the phosphinothricin sensitivephenotype. Five individuals were found to be sensitive tophosphinothricin.

[0351] Genomic DNA was isolated and analyzed by Southern blot analysis.The blot was hybridized with probes for the bt, bar and amp genes.Results of this analysis are shown in Table 9. TABLE 9 Summary of DBT418Recombinants Displaying Phosphinothricin Sensitivity Phenotypes andGenotypes # full- # partial # full- # full- Individual length bar bargene length Bt length Amp Row Plant Phenotype^(a) gene copies copiesgene copies copies 03 09 S 0 1 1 1 08 17 S 0 1 >3 >5 09 07 S 0 1 2 2 1118 S 0 1 2 2 15 11 S 0 1 2 2 Normal 1 1 2 3 DBT418 R

[0352] All five phosphinothricin-sensitive individuals lacked the fulllength bar gene present in phosphinothricin-resistant DBT418. The dataalso showed that each phosphinothricin-sensitive plant still containedtransgene DNA corresponding to the partial bar gene copy, the Bt geneand the amp gene. The copy number of these transgenes varied among thephosphinothricin sensitive individuals. The data shows three classes ofvariants. Plant 03-09 lacked the full length bar gene copy, but retaineda partial bar gene copy, one copy of the Bt gene and one copy of the ampgene (FIG. 10). Plants 09-07, 11-18, and 15-11 represent a second classof variants that lacked the full length bar gene, but retained a partialbar gene, two copies of the Bt gene, and two copies of the amp gene(FIG. 10). Finally, a third class was observed where the full length bargene copy was absent, but a partial bar gene copy was retained, andwhere the copy number of the Bt gene and amp gene were increasedcompared to DBT418.

EXAMPLE 2 Deletion of nptII or cryIA(b) Gene from the Transgenic Events“MON849” and “MON850”

[0353] Transformation events (MON849) and (MON 850) were produced bymicroprojectile bombardment of cells with plasmid vector using pMON19344(FIG. 4). The structure of the MON849 transgene insert is diagramed inFIG. 11. The insert has one copy of an nptII gene conferring resistanceto kanamycin and one copy of a cryIA(b) Bt gene conferring resistance tocertain insect pests. Both the nptII and cryIA(b) coding regions areflanked on the 5¢ ends by identical 35S promoters and hsp70 introns.Both the nptII and cryIA(b) coding regions are flanked on the 3¢ ends byidentical nos terminators. Recombination events between the 35S promoterand hsp70 intron regions of the cryIA(b) gene and the 35S promoter andhsp70 intron regions of the nptII gene result in the loss of thecryIA(b) gene (FIG. 11). Recombination events between the nos terminatorregion of the cryIA(b) gene and the nos terminator region of the nptIIgene result in the loss of the nptII gene (FIG. 11). The latterrecombination event is useful in that (i) the resultant plant would begenetically more stable, as loss of the cryIA(b) gene would not occurduring seed increase, (ii) the resultant plant would be phenotypicallymore stable, as there would be no repeated genetic elements within theinsert, and (iii) the ancillary DNA sequence encoding nptII that doesnot contribute to the designed insect resistance phenotype is deleted.

[0354] Plant material was prepared by self pollinating plants hemizygousfor the transgene insert, identifying individuals homozygous for thetransgene insert in the subsequent generation, and crossing thehomozygous individuals to nontransgenic plants. The resulting populationwas hemizygous for the transgene insert.

[0355] To identify non-reciprocal recombinants within this MON849progeny population, transgene expression assays were carried out onapproximately 1,000 individuals and 7 individuals that differed inphenotype from the parent were identified (Table 10) (frequency of0.4%). PCR analysis carried out for the cryIA(b) and nptII genes showedthat the lack of a transgene phenotype correlated with the absence ofthe particular transgene. Plant 20-102-A (plant numbers refer torange-row-stake number, as listed in Table 10) appears to be arecombinant that has lost the nptII gene. Plant 20-103-3 lacks bothtransgenes and may be the result of pollen contamination. Five MON849progeny plants show an apparent recombination in which the cryIA(b) genewas lost and the nptII gene retained. A similar transgene stabilityassay was also carried out on approximately 1,000 individuals derivedfrom a parent plant that was homozygous for the MON850 event and about0.7% of the individuals differed from the parent. One MON849 progenyplant and one MON850 progeny plant showed an apparent recombination inwhich the nptII gene was lost and the cryIA(b) gene retained. Therecombinant individuals lacking the nptII gene were crossed with avariety of inbreds. TABLE 10 Genetic Analysis (PCR) of Mon849 and Mon850Plants Displaying Off-type Phenotypes Phenotypes Genotypes (PCR) EventRange Row Stake # CrylA(b) NPTII CrylA(b) NPTII MON850 19 126 8¹ o o o oMON850 18 125 7¹ o o o o MON850 18 129 B + o + o MON850 19 125 C + o + +MON849 20 102 A + o + o MON849 18 113 6 o + o + MON849 20 105 5 o + o +MON849 19 105 4 o + o + MON849 20 103 3 o o o o MON849 20  99 2 o + o +MON849 19  99 1 o + o o

[0356] Southern blot analyses of the recombinant MON849 individuals werecarried out in order to confirm that gene deletion was mediated byhomologous recombination. As shown in Table 11, both nptII+/cryIA(b)−and nptII−/cryIA(b)+ individuals displayed a pattern of hybridizingbands that are indicative of homologous recombination mediated transgenedeletion. TABLE 11 Southern hybridization band sizes for MON849 F1derivatives Probe A = Phenotype CrylA(b) Probe B = nptII CrylA(b) % inF₁ EcoRI NcoI EcoRI (E) - Kan^(R) ELISA progeny^(*) (E) (N) NcoI (N)XbaI (X) + + 99.3% 10.0 6.1 2.6 5.2 + ∘  0.6% ? ? 7.3 5.2 ∘ +  0.1% 10.05.9 ? ?

[0357] Quantitative ELISA analysis of a nptII−/cryIA(b)+ individualderived from both MON849 and MON850 events indicated that deletion ofthe nptII gene did not significantly compromise the expression of thecryIA(b) gene as shown in Table 12. TABLE 12 Quantitative ELISA onMON849 and MON850 F1 Derivatives Phenotype Protein (μg/g dry wt.)Kan^(R) CrylA(b) MON849 MON850 + + 18.48 11.22 ∘ + 11.59 17.36

[0358] Finally, in looking at the relationship between the repeatedsequences flanking the deleted gene and the frequency of recombination,a direct correlation was observed between the length of the directrepeat sequences flanking the deleted gene and the observed frequency ofhomologous recombination mediated transgene deletion (Table 13). Theobserved gene deletion frequency is estimated at about 0.1% per 287 bpof homologous direct repeat sequence ±19 bp (S.E). TABLE 13 CorrelationBetween Flanking Direct Repeat Length and Frequency of Intervening GeneDeletion Deleted Direct Repeat % Deletion Event Gene Repeats LengthRecombinants Mon849 nptII nos 0.3 kbp 0.1% Mon849 CrylA(b) e35S-hsp701.5 kbp 0.6% DBT418 bar pDPG354 6.2 kbp 2.0%

EXAMPLE 3 Alteration of a Transgene Insertion Event in Transformed Cells

[0359] The plasmid vector pMON36133 (FIG. 12) was constructed wherein aneomycin phosphotransferase II (nptII) gene is flanked on both the 5′and 3′ ends by direct repeats of sequences derived from the 3′ end ofthe maize hsp70 intron. The vector further comprises a gene encodinggreen fluorescent protein (GFP) that lacks a promoter and is notexpressed in a plant cell. Deletion of the sequences between therepeated hsp70 sequences produces a transgene wherein the 35S promoterand hsp70 intron are operable linked to the GFP gene and therefore, theGFP protein is expressed.

[0360] The plasmid vector pMON36133 was introduced into Black MexicanSweet maize cells using microprojectile bombardment. Transformed calluswas selected based on resistance to kanamycin conferred by the nptIIgene. Sectors of GFP expressing tissues were observed in thetransformants, indicating that the nptII gene was deleted, therebyactivating expression of the GFP gene.

[0361] In conclusion, homologous recombination can be used to removeunwanted transgenic DNA sequences from genetically transformed plants.Target trait gene expression was not compromised by the deletion of alinked marker gene. Moreover, the observed recombination frequencyappears to be directly proportional to the length of the repeats withinthe region being targeted for gene deletion. Thus, transformation can bedesigned to facilitate subsequent gene deletion, such as in pMON19344.

[0362] All publications, patents and patent applications cited above areincorporated by reference herein, as though fully set forth. Theinvention has been described with reference to various specific andpreferred embodiments and will be further described by reference to thefollowing detailed examples. It is understood, however, that there aremany extensions, variations, and modifications on the basic theme of thepresent invention beyond that shown in the examples and description,which are within the spirit and scope of the present invention.

REFERENCES

[0363] The references listed below are incorporated herein by referenceto the extent that they supplement, explain, provide a background for,or teach methodology, techniques, and/or compositions employed herein.

[0364] U.S. Pat. No. 4,535,060

[0365] U.S. Pat. No. 4,940,835

[0366] U.S. Pat. No. 4,940,838

[0367] U.S. Pat. No. 4,959,317

[0368] U.S. Pat. No. 5,134,074

[0369] U.S. Pat. No. 5,168,053

[0370] U.S. Pat. No. 5,254,801

[0371] U.S. Pat. No. 5,258,300

[0372] U.S. Pat. No. 5,268,526

[0373] U.S. Pat. No. 5,276,268

[0374] U.S. Pat. No. 5,290,924

[0375] U.S. Pat. No. 5,302,523

[0376] U.S. Pat. No. 5,322,783

[0377] U.S. Pat. No. 5,354,855

[0378] U.S. Pat. No. 5,384,253

[0379] U.S. Pat. No. 5,451,513

[0380] U.S. Pat. No. 5,464,765

[0381] U.S. Pat. No. 5,482,852

[0382] U.S. Pat. No. 5,489,520

[0383] U.S. Pat. No. 5,500,365

[0384] U.S. Pat. No. 5,508,184

[0385] U.S. Pat. No. 5,508,468

[0386] U.S. Pat. No. 5,510,471

[0387] U.S. Pat. No. 5,538,877

[0388] U.S. Pat. No. 5,538,880

[0389] U.S. Pat. No. 5,550,318

[0390] U.S. Pat. No. 5,563,055

[0391] U.S. Pat. No. 5,563,324

[0392] U.S. Pat. No. 5,590,390

[0393] U.S. Pat. No. 5,591,616

[0394] U.S. Pat. No. 5,593,963

[0395] U.S. Pat. No. 5,610,042

[0396] U.S. Pat. No. 5,624,824

[0397] U.S. Pat. No. 5,625,047

[0398] U.S. Pat. No. 5,654,182

[0399] U.S. Pat. No. 5,658,772

[0400] U.S. Pat. No. 5,689,052

[0401] U.S. Pat. No. 5,723,765

[0402] U.S. Pat. No. 5,743,477

[0403] U.S. Pat. No. 5,780,708

[0404] U.S. Pat. No. 5,780,709

[0405] U.S. Pat. No. 5,792,924

[0406] U.S. Pat. No. 5,801,030

[0407] U.S. Pat. No. 5,831,011

[0408] U.S. Pat. No. 6,627,061

[0409] EP 0154204 B1

[0410] PCT Publication No. WO 92/17598

[0411] PCT Publication No. WO 97/04103

[0412] PCT Publication No. WO 97/26366

[0413] PCT Publication No. WO 98/26064

[0414] PCT Publication No. WO 99/32642

[0415] Abdullah et al., 1986. Biotechnology, 4:1087

[0416] Abel, P. P., Nelson, R. S., De, B., Hoffman, N., Rogers, S. G.,Fraley, R. T. and

[0417] Beachy, R. N. 1986. Science 232:738-743.

[0418] Abremski et al. 1983. Cell 32:1301

[0419] An, G. et al. 1989. Plant Cell 1:115-122.

[0420] Armaleo et al., 1990. Curr. Genet. 17(2):97-103

[0421] Assad, F. A., Signer, E. R., 1992, Genetics 132:553-566.

[0422] Athma, P., Peterson, T., 1991. Genetics 128:163-173

[0423] Barkai-Golan, R., Mirelman, D., Sharon, N. 1978. Arch. Microbiol116:119-124.

[0424] Bates, Mol. Biotechnol., 1994. 2(2):135-145

[0425] Battraw and Hall, 1991. Theor. App. Genet., 82(2):161-168

[0426] Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. & Schaller, H.1982. Gene 19(3):327-36.8

[0427] Bernal-Lugo, I. and Leopold, A. C. 1992. Plant Physiol.98:1207-1210.

[0428] Berzal-Herranz et al., 1992. Genes and Devel., 6:129-134

[0429] Bevan, M., Barnes, W. M., Chilton, M. D., 1983. Nucleic AcidResearch. 11:369-385.

[0430] Bhattacharjee; An; Gupta, 1997. J. Plant Bioch. and Biotech. 6,(2):69-73

[0431] Blackman, S. A., Obendorf, R. L., Leopold, A. C. 1992. PlantPhysiol. 100:225-230.

[0432] Bol, J. F., Linthorst, H. J. M., Cornelissen, B. J. C. 1990.Annu. Rev. Phytopath. 28:113-138.

[0433] Bottjer et al., 1985. Experimental Parasitology, 60:239-244

[0434] Bouchez D., Tokuhisa J. G., Llewellyn D. J., Dennis E. S. andEllis J. G., 1989. EMBO Journal 8(13):4197-4204.

[0435] Bower et al., 1992. The Plant Journal, 2:409-416.

[0436] Bowler, C., Van Montagu, M., and Inze, D. 1992. Ann Rev. PlantPhysiol. 43:83-116.

[0437] Branson, T. F. and Guss, P. L. 1972. Proceedings North CentralBranch Entomological Society of America 27:91-95.

[0438] Broakaert, W. F., Parijs, J., Leyns, F., Joos, H., Peumans, W. J.1989. Science 245:1100-1102.

[0439] Buchanan-Wollaston et al., 1992. Plant Cell Reports 11:627-631.

[0440] Buising and Benbow, 1994. Mol Gen Genet, 243(1):71-81.

[0441] Callis, J., Fromm, M., Walbot, V. 1987., Genes and Develop.1:1183-1200.

[0442] Campbell, W. C. ed. 1989. In: Avermectin and Abamectin.

[0443] Carrer, H., et al. 1993. Mol. Gen. Genet. 241:49

[0444] Casa et al., 1993. Proc. Nat'l Acad. Sci. USA, 90(23):11212-11216

[0445] Cech et al., 1981. Cell, 27:487-496

[0446] Chandler, V. L., Radicella, J. P., Robbins, P. P., Chen, J.,Turks, D. 1989. The Plant Cell 1:1175-1183

[0447] Chowrira et al, 1993. J Biol Chem., 268:19458-62

[0448] Chowrira et al., 1994. J Biol. Chem., 269:25856-25864

[0449] Christou; Murphy; Swain, 1987. Proc. Nat'l Acad. Sci. USA,84(12):3962-3966

[0450] Chu C. C., Wang C. C., Sun C. S., Hsu C., Yin K. C., Chu C. Y.,Bi F. Y. 1975. Scientia Sinica 18:659-668

[0451] Clark, R. 1982. J Plant Nutrition 5:1039.

[0452] Coe, E. H., Neuffer, M. G., and Hoisington, D. A. 1988. in Cornand Corn Improvement, Sprague, G. F. and Dudley, J. W., eds., pp. 81-258

[0453] Comai et al., 1985. Nature, 317:741-744

[0454] Conkling, M. A., Cheng, C. L., Yamamoto, Y. T., Goodman, H. M.1990. Plant Physiol. 93:1203-1211

[0455] Coruzzi et al. 1971. EMBO J. 3:1671

[0456] Coxson, D. S., McIntyre, D. D., and Vogel, H. J. 1992. Biotropica24:121-133.

[0457] Cuozzo, M., O'Connell, K. M., Kaniewski, W., Fang, R. X., Chua,N. and Turner, N. 1988. Bio/Technology 6:549-553.

[0458] Cutler, A. J., Saleem, M., Kendell, E., Gusta, L. V., Georges,F., Fletcher, G. L. 1989. J Plant Physiol 135:351-354.

[0459] Czako, M. and Marton, L. 1994. The herpes simplex virus thymidinekinase gene as a conditional negative selectable marker gene inArabidopsis thaliana. Plant Physiol. 104: 1067-1071.

[0460] Czapla and Lang 1990. J. Econ. Entomol. 83:2480-2485.

[0461] Davies, T. G. E., Thomas, H., Thomas, B., Rogers, L. J. 1990.Plant Physiol. 93:588-595.

[0462] De Block et al., 1987. The EMBO Journal, 6(9):2513-2518

[0463] De Block, De Brouwer, Tenning, 1989. Plant Physiol., 91:694-701

[0464] Dekeyser et al. 1990. Plant Cell 2:591-602.

[0465] Dellaporta, S., Greenblatt, I., Kermicle, J., Hicks, J. B.,Wessler, S. 1988. in Chromosome Structure and Function: Impact of NewConcepts, 18th Stadler Genetics Symposium, J. P. Gustafson and R.Appels, eds (New York: Plenum Press) pp. 263-282.

[0466] Depicker et al., 1988. Plant Cell Reports, 7:63-66

[0467] D'Halluin et al., 1992. Plant Cell, 4(12):1495-1505

[0468] Dotson, S. B., Lnahan, M. B., Smith, A. G., and Kishore, G. M.1996a. A phosphonate monoester hydrolase from Burkholderia caryophilliPG2982 is useful as a conditional lethal gene in plants. Plant J. 10:383-392.

[0469] Dotson, S. B., Smith, C. E., Ling, C. S., Barry, G. F. andKishore, G. M. 1996b. J. Biol.

[0470] Chem. 271 (42): 25754-25761.

[0471] Dure, L., Crouch, M., Harada, J., Ho, T. -H. D., Mundy, J.,Quatrano, R., Thomas, T., and Sung, Z. R. 1989. Plant Molecular Biology12:475-486.

[0472] Ebert, P. R., Ha, S. B., An. G. 1987. PNAS 84:5745-5749.

[0473] Ehrenshaft et al., 1999. Current Genetics, 34(6):478-485

[0474] Ellis J. G., Llewellyn D. J., Walker J. C., Dennis E. S., andPeacock W. J., 1987. EMBO Journal 6(11):3203-3208.

[0475] Erdmann, N., Fulda, S., and Hagemann, M. 1992. J. Gen.Microbiology 138:363-368.

[0476] Evans, D. A. and Paddock, E. F. 1979. Mitotic Crossing-Over inHigher Plants. In: Plant Cell and Tissue Culture, Principles andApplications. Sharp, W. R. et al. eds. Ohio State Unversity Press.

[0477] Finkle B. J., Ulrich J. M., Rains W., Savarek S. J., 1985. PlantSci 42:133-140.

[0478] Fitzpatrick, T. 1993. Gen. Engineering News 22 (March 7):7.

[0479] Forster and Symons, 1987. Cell, 49:211-220

[0480] Fraley et al., 1985. Bio/Technology, 3:629-635

[0481] Fransz, P. F., de Ruijter, N. C. A., Schel, J. H. N. 1989. PlantCell Rep 8:67-70

[0482] Fromm et al., 1986. Nature 319:791-793

[0483] Fromm M. E. et al. 1990. Bio/Technology, 8, 833

[0484] Fromm, H., Katagiri, F., Chua, N. H. 1989. The Plant Cell1:977-984.

[0485] Gal, S., Pisan, B., Hohn, T., Grimsley, N., Hohn, B., 1991, EMBOJ. 10:1571-1578

[0486] Gallie, D. R., Lucas, W. J., Walbot, V. 1989. The Plant Cell1:301-311.

[0487] Gatehouse, A. M., Dewey, F. M., Dove, J., Fenton, K. A., Dusztai,A. 1984. J Sci Food Agric 35:373-380.

[0488] Gelvin, S. B., Schilperoort, R. A., Varma, D. P. S., eds., 1990.Plant Molecular Biology Manual

[0489] Gerlach et al., 1987. Nature 328:802-805

[0490] Ghosh-Biswas et al., 1994. J. Biotechnol., 32(1):1-10

[0491] Gordon-Kamm W. J. et al. 1990. Plant Cell, 2, 603

[0492] Goring, D. R., Thomson, L., Rothstein, S. J. 1991. Proc. Natl.Acad Sci. USA 88:1770-1774.

[0493] Guerrero, F. D., Jones, J. T., Mullet, J. E. 1990. PlantMolecular Biology 15:11-26.

[0494] Gupta, A. S., Heinen, J. L., Holaday, A. S., Burke, J. J., andAllen, R. D. 1993. Proc. Natl. Acad. Sci USA 90:1629-1633.

[0495] Hagio, Blowers, Earle, 1991. Plant Cell Rep., 10(5):260-264

[0496] Hallauer et al. 1988. In: Corn and Corn Improvement, Sprague etal. (eds.) pp. 463-564

[0497] Hammock, B. D., Bonning, B. C., Possee, R. D., Hanzlik, T. N.,and Maeda, S. 1990. Nature 344:458-461.

[0498] Hardy et al. 1997. J. Virology 71:1842

[0499] Haseloff and Gerlach, 1988. Nature, 334:585-591

[0500] He et al, 1994. Plant Cell Reports, 14 (2-3):192-196

[0501] Heijne et al. 1989. Eur. J. Biochem., 180, 535

[0502] Hemenway, C., Fang, R., Kaniewski, W. K., Chua, N. and Turner, N.E. 1988. The EMBO J. 7:1273-1280.

[0503] Hensgens et al., 1993. Plant Mol. Biol., 22(6):1101-1127

[0504] Herrera-Estrella et al. 1990. Proc. Natl. Acad. Sci. USA,87:9534-9537.

[0505] Hiei et al., 1997. Plant. Mol. Biol., 35(1-2):205-218

[0506] Hilder, V. A., Gatehouse, A. M. R., Sheerman, S. E., Barker, R.F., Boulter, D. 1987. Nature 330:160-163.

[0507] Hinchee, M. A. W., Connor-Ward, D. V., Newell, C. A., McDonell,R. E., Sato, S. J.,

[0508] Gasser, C. S., Fischhoff, D. A., Re, C. B., Fraley, R. T.,Horsch, R. B. 1988. Bio/technol 6:915-922.

[0509] Holmberg et al., 1997. Nature Biotechnology, 15(3):244-247

[0510] Hou and Lin, 1996. Plant Physiology, 111 (Sup 2):166

[0511] Hudspeth, R. L. and J. W. Grula. 1989. Plant Mol. Biol.12:579-589.

[0512] Ikeda, H., Kotaki, H., Omura, S. 1987. J Bacteriol 169:5615-5621.

[0513] Ikuta, N., Souza, M. B. N., Valencia, F. F., Castro, M. E. B.,Schenberg, A. C. G., Pizzirani-Kleiner, A., Astolfi-Filho, S. 1990.Bio/technol 8:241-242.

[0514] Ishida et al., Nat. Biotechnol., 1996. 14(6):745-750

[0515] Jefferson R. A., 1987. Plant Molecular Biology Reporter, 5,387-405

[0516] Jelesko, J. G., Harper, R., Furuya, M., Gruissem, W., Proc. Natl.Acad. Sci USA 96:10302-10307

[0517] Jenkins, G., and Cundliffe, E. 1991. Gene 108(1):55-62.

[0518] Johnson et al., 1989. Proc. Natl. Acad. Sci. USA, 86:9871-9875

[0519] Joshi, C. P. 1987. Nucleic Acids Res., 15:6643-6653.

[0520] Joyce, 1989. Nature, 338:217-244

[0521] Kaasen, I., Falkenberg, P., Styrvold, O. B., Strom, A. R. 1992.J. Bacteriology 174:889-898.

[0522] Kaeppler et al., 1990. Plant Cell Reports 9: 415-418

[0523] Kaeppler, Somers, Rines, Cockburn, 1992. Theor. Appl. Genet.,84(5-6):560-566

[0524] Karsten, U., West, J. A. and Zuccarello, G. 1992. Botanica Marina35:11-19.

[0525] Kasuga, et al. 1999. Nat. Biotech. 17:287

[0526] Katz et al. 1983. J. Gen. Microbiol. 129:2703-2714.

[0527] Keegstra et al. 1989. Ann. Rev. Plant Physiol. Plant Mol. Biol.,40:471

[0528] Keller et al. 1989. EMBO J., 8(5):1309-1314.

[0529] Kim and Cech, 1987. Proc. Natl. Acad. Sci. USA, 84:8788-8792

[0530] Klee, Yanofsky, Nester, 1985. Bio-Technology, 3(7):637-642

[0531] Knittel, Gruber; Hahne; Lenee, 1994. Plant Cell Reports,14(2-3):81-86

[0532] Koster, K. L. and Leopold, A. C. 1988. Plant Physiol. 88:829-832.

[0533] Laufs, J., Wirtz, U., Kammann, M., Matzeit, V., Schaefer, S.,Schell, J., Czernilofsky, A. P., Baker, B., and Gronenborn, B. 1990.Proc. Natl. Acad. Sci USA. 87:7752-7756.

[0534] Laursen, C. M., Krzyzek, R. A., Flick, E. E., Anderson, P. C.,Spencer, T. M. 1994. Plant Molecular Biology. 24:51

[0535] Lawton, M. A., Tierney, M. A., Nakamura, I., Anderson, E.,Komeda, Y., Dube, P., Hoffman, N., Fraley, R. T., Beachy, R. N. 1987.Plant Mol. Biol. 9:315-324.

[0536] Lazzeri, 1995. Methods Mol. Biol., 49:95-106

[0537] Lee, C. A. and Saier, M. H. Jr. 1983. J. of Bacteriol.153:685-692.

[0538] Lee; Suh; Lee, 1989. Korean J. Genet., 11(2):65-72

[0539] Levings, C. S., III. 1990. Science 250:942-947

[0540] Lichtenstein, C., Paszkowski, J., Hohn, B., in: HomologousRecombination and Gene Silencing in Plants, Ed. J. Paszkowski. Kluwer,Dordrecht, The Netherlands, 1994

[0541] Lieber and Strauss, 1995. Mol. Cell. Biol., 15: 540-551

[0542] Loomis, S. H., Carpenter, J. F., Anchordoguy, T. J., Crowe, J.H., and Branchini, B. R. 1989. J. Expt. Zoology 252:9-15.

[0543] Lorz et al., 1985. Mol Gen Genet, 199:178-182

[0544] Luo H., Lyznik, L. A., Gidoni D., Hodges, T. K. 2000. Plant J.Aug;23(3):423-430

[0545] Lyznik, L. A., Rao, K. V., Hodges, T. K. 1996. Nucleic Acids Res,Oct 1; 24(19):3784-9

[0546] Marcotte et al., 1988. Nature, 335:454-457.

[0547] Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J. andGoldberg, R. B. 1990. Nature 347:737-741.

[0548] McCabe, Martinell, 1993. Bio-Technology, 11(5):596-598

[0549] McCormac et al., 1998. Euphytica, v. 99 (1) p. 17-25

[0550] McElroy et al 1990. Plant Cell, 2:163

[0551] McElroy et al. 1991. Molec. Gen. Genet., 231:150-160 Methods inEnzymology, 153, 292, 1987.

[0552] Michel and Westhof, 1990. J Mol. Biol., 216:585-610

[0553] Moll, B., et al. 1990. Mol. Gen. Genet. 221:245

[0554] Mundy, J. and Chua, N. -H. 1988. The EMBO J. 7:2279-2286.

[0555] Murakami T., Anzai H., Imai S., Satoh A., Nagaoka K., Thompson C.J. 1986. Mol Gen Genet 205:42-50.

[0556] Murashige T., Skoog F. 1962. Physiol Plant 15:473-497.

[0557] Murdock et al. 1990. Phytochemistry 29:85-89.

[0558] Nagatani, Honda, Shimada, Kobayashi, 1997. Biotech. Tech.,11(7):471-473

[0559] Napoli, Lemieux, Jorgensen, 1990. Plant Cell, 2:279-289

[0560] Niedz et al. 1995. Plant Cell Reports, 14:403

[0561] Odell, J. T., Nagy, F., Chua, N. H. 1985. Nature 313:810-812.

[0562] Ogawa et al., 1973. Sci. Rep., 13:42-48

[0563] Omirulleh et al., 1993. Plant Mol. Biol., 21(3):415-428

[0564] Ow, D. W., Wood, K. V., DeLuca, M., deWet, J. R., Helinski, D.R., Howell, S. H. 1986. Science 234:856-859.

[0565] Palukaitis et al., 1979. Virology, 99:145-151

[0566] Perlak F. J., Fuchs R. L., Dean D. A., McPherson S. L., andFischhoff D. A., 1991. Proc. Natl. Acad. Sci. USA 88:3324-3328.

[0567] Perrimanetal., 1992. Gene, 113:157-163

[0568] Peterhans, A., Schlupmann, H., Basse, C., Paxzkowski, J., 1990,EMBO J. 9: 3437-45Phi-Van et al., 1990. Mol. Cell. Biol., 10:2302-2307

[0569] Piatkowski, D., Schneider, K., Salamini, F. and Bartels, D. 1990.Plant Physiol. 94:1682-1688.

[0570] Potrykus 1. 1989. Trends Biotechnol 7:269-273.

[0571] Potrykus, I., Saul, M. W., Petruska, J., Paszkowski, J.,Shillito, R. D. 1985. Mol Gen Genet 199:183-188

[0572] Prasher et al., 1985. Biochem. Biophys. Res. Commun.,126(3):1259-1268

[0573] Prasher, et al. 1985. Biochem. Biophys. Res. Commun., 126(3):1259-1268.

[0574] Prody et al., 1986. Science, 231:1577-1580

[0575] Reed, R. H., Richardson, D. L., Warr, S. R. C., Stewart, W. D. P.1984. J. Gen. Microbiology 130:1-4.

[0576] Reinhold-Hurek and Shub, 1992. Nature, 357:173-176

[0577] Reiss, B., Klemm, M., Kosak, H., Schell, J., 1996. Proc. Natl.Acad. Sci. USA 93:3094-3098

[0578] Rensburg et al., 1993. J. Plant Physiol., 141:188-194

[0579] Rhodes et al., 1995. Methods Mol. Biol., 55:121-131

[0580] Ritala et al., 1994. Plant Mol. Biol., 24(2):317-325 Robbins, T.P., Walker, E. L.,

[0581] Kermicle, J. L., Alleman, M., Dellaporta, S. L. 1991. Genetics129: 271-283 Rogers

[0582] et al., 1987. Methods Enzymol., 153:253-277

[0583] Sambrook, J., Fritsch, E. F., and Maniatus, T. 1989. MolecularCloning, A Laboratory Manual 2nd ed. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.

[0584] Sengupta-Gopalan, 1985. Proc. Natl. Acad. Sci. USA, 82:3320

[0585] Shagan, T., Bar-Zvi, D. 1993. Plant Physiol. 101:1397-1398

[0586] Shalev, G., Sitrit, Y., Avivi-Ragolski, N., Lichtenstein, C.,Levy, A. 1999. Proc. Natl Acad. Sci. USA 96: 7398-7402.Shapiro, In:Mobile Genetic Elements, 1983.

[0587] Singsit et al., 1997. Transgenic Res., 6(2):169-176

[0588] Skriver, K. and Mundy, J. 1990. Plant Cell 2:503-512.

[0589] Smith, Watson, Bird, Ray, Schuch, Grierson, 1990. Mol. Gen.Genet., 224:447-481

[0590] Spencer, T. M., O'Brien, J. V., Start, W. G., Adams, T. R.,Gordon-Kamm, W. J. and Lemaux, P. G. 1992. Plant Molecular Biology18:201-210.

[0591] Stalker, D. M., McBride, K. E., and Malyj, L. 1988. Science 242:419-422

[0592] Stemberg et al. 1981. Cold Spring Harbor Symposia on QuantitativeBiology, Vol. XLV 297

[0593] Stief et al, 1989. Nature 341:343

[0594] Stiefel et al. 1990. The Plant Cell, 2:785-793.

[0595] Stouggard, J. 1993. The Plant Journal 3: 755-761.

[0596] Sullivan, Christensen, Quail, 1989. Mol. Gen. Genet.,215(3):431-440

[0597] Sutcliffe, J. G. 1978. Proc Natl Acad Sci USA 75:3737-3741

[0598] Swoboda, P., Hohn, B., Gal, S., 1993, Mol. Gen. Genet. 237:33-40

[0599] Swoboda, P., Gal, S., Hohn B., Puchta, H., 1994, Intrachromosomalhomologous recombination in whole plants. EMBO J. 13:484-489

[0600] Symons, R. H. 1981. Nucleic Acids Res 9(23):6527-37.

[0601] Symons, R. H. 1992. Annu. Rev. Biochem., 61:641-671

[0602] Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J., Stahl, F.W., 1983, The double-strand break repair model for recomgination. Cell33:25-35

[0603] Tarczynski, M. C., Jensen, R. G., and Bohnert, H. J. 1993.Science 259:508-510.

[0604] Tarczynski, M. C., Jensen, R. G., and Bohnert, H. J. 1992. Proc.Natl. Acad. Sci. USA, 89: 2600

[0605] Thillet, J., Absil, J., Stone, S. R., Pictet, R. 1988. J BiolChem 263:12500-12508.

[0606] Thompson et al, 1995. Euphytica, 85(1-3):75-80

[0607] Thompson et al., 1987. The EMBO Journal, 6(9):2519-2523

[0608] Tingay et al., 1997. The Plant Journal v. 11 (6) p.1369-1376

[0609] Tomes et al., 1990. Plant. Mol. Biol. 14(2):261-268

[0610] Torbet, Rines, Somers, 1998. Crop Science, 38(1):226-231

[0611] Torbet, Rines, Somers, 1995. Plant Cell Reports, 14(10):635-640

[0612] Toriyama et al., 1986. Theor Appl. Genet., 73:16

[0613] Tovar and Lichtenstein, 1992. The Plant Cell 4: 319-332.

[0614] Tsukada; Kusano; Kitagawa, 1989. Plant Cell Physiol.,30(4)599-604

[0615] Twell D., Klein T. M., Fromm M. E., McCormick S. 1989. PlantPhysiol 91:1270-1274.

[0616] Uchimiya et al., 1986. Mol. Gen. Genet., 204:204-207.

[0617] Ugaki et al. 1991. Nucl Acid Res, 19:371-377.

[0618] Van der Krol et al., 1990. Plant Cell, 2:291-99

[0619] Van Eck; Blowers; Earle, 1995. Plant Cell Reports, 14(5):299-304

[0620] Vasil, V., Clancy, M., Ferl, R. J., Vasil, I. K., Hannah, L. C.1989. Plant Physiol. 91:1575-1579.

[0621] Viet, B., Vollbrecht, E., Mathem, J., Hake, S. 1990. Genetics125: 623-631.Vernon, D. M. and Bohnert, H. J. 1992. The EMBOJ.11:2077-2085.

[0622] Walker, J. C., Howard, E. A., Dennis, E. S., Peacock, W. J 1987.Proc. Natl. Acad. Sci. USA 84:6624-6628.

[0623] Wang, Y., Zhang, W., Cao, J., McEhoy, D. and Ray Wu. 1992.Molecular and Cellular Biology 12:3399-3406.

[0624] Watrud et al, In: Engineered Organisms and the Environment, 1985.

[0625] Withers L. A., King P. J. 1979. Plant Physiol 64:675-678.

[0626] Wolter, F., Schmidt, R., and Heinz, E. 1992. The EMBO J.11:4685-4692.

[0627] Xiang and Guerra, Plant Physiol., 1993. 102:287-293

[0628] Xu et al., Plant Physiol., 1996. 110:249-257

[0629] Yamada et al, 1986. Plant Cell Rep., 4:85

[0630] Yamaguchi-Shinozaki, K., Koizumi, M., Urao, S., Shinozaki, K.1992. Plant Cell Physiol. 33:217-224.

[0631] Yang, N. S., Russell, D. 1990. Proc. Natl. Acad. Sci. USA87:4144-4148.

[0632] Yuan and Altnan, 1994. Science, 263:1269-1273

[0633] Yuan et al., 1992. Proc. Natl. Acad. Sci. USA, 89:8006-8010

[0634] Zhang et al., 1997. Mol Biotechnol., 8:223-31

[0635] Zheng and Edwards, 1990. J Gen. Virol., 71:1865-1868

[0636] Zhou et al., 1993. Plant Cell Reports, 12(11). 612-616

[0637] Zubko, E., Scutt, C., Meyer, P., 2000,. Nature Biotechnology18:442-445

What is claimed is:
 1. A method of preparing a transgenic cell having analtered transgene insertion comprising: a) obtaining a first transgeniccell, wherein the transgene insertion DNA sequence comprises apre-selected DNA sequence flanked by directly repeated DNA sequences; b)obtaining a plurality of progeny cells of any generation of the firsttransgenic cell; c) selecting a progeny transgenic cell wherein at leasta portion of the transgene insertion is altered as compared to the firsttransgenic cell.
 2. The method of claim 1 , wherein said transgenic cellis a plant cell.
 3. The method of claim 1 , wherein said transgenic cellis homozygous for the transgene insertion DNA sequence.
 4. The method ofclaim 1 wherein the pre-selected DNA sequence comprises a selectablemarker gene or a reporter gene.
 5. The method of claim 1 wherein thepre-selected DNA sequence further comprises a negative selectable markergene.
 6. The method of claim 5 wherein said negative selectable markergene comprises a pehA gene.
 7. The method of claim 1 wherein thepre-selected DNA sequence comprises a bar, nptII, EPSPS, GFP or cryIA(b)gene.
 8. A method of preparing a fertile transgenic plant having analtered transgene insertion comprising regenerating a plant from theprogeny transgenic cell of claim 1 .
 9. A method of preparing a fertiletransgenic plant having an altered transgene insertion comprising: a)obtaining a first transgenic plant, wherein the transgene insertion DNAsequence comprises a pre-selected DNA sequence flanked by directlyrepeated DNA sequences; b) obtaining a plurality of progeny of anygeneration of the first transgenic plant; c) selecting a progeny fertiletransgenic plant wherein at least a portion of the transgene insertionis altered as compared to the first fertile transgenic plant.
 10. Themethod of claim 9 , wherein said transgenic plant is homozygous for thetransgene insertion DNA sequence.
 11. The method of claim 9 wherein thepre-selected DNA sequence comprises a selectable marker gene or areporter gene.
 12. The method of claim 9 wherein the pre-selected DNAsequence comprises a bar, nptII, EPSPS or cryIA(b) gene.
 13. The methodof claim 9 wherein the plurality of progeny plants are obtained byself-pollination.
 14. The method of claim 9 wherein the plurality ofprogeny plants are obtained by outcrossing to produce hybrid progeny.15. The method of claim 9 wherein the plurality of progeny plants areobtained by inbreeding to produce inbred plants.
 16. The method of claim9 wherein the plant is a monocot plant.
 17. The method of claim 16wherein the monocot plant is a maize, barley, sorghum, wheat, rye orrice plant.
 18. The method of claim 17 wherein the plant is a maizeplant.
 19. The method of claim 9 wherein the plant is a dicot plant. 20.The method of claim 19 wherein the dicot plant is a soybean, cotton,canola or potato plant.
 21. The method of claim 9 wherein at least aportion of the transgene insertion is altered in that it has beendeleted, amplified, or rearranged.
 22. A fertile transgenic plantproduced by the method of claim 9 .
 23. The fertile transgenic plant ofclaim 22 which is inbred.
 24. The fertile transgenic plant of claim 22which is hybrid.
 25. The fertile transgenic plant of claim 22 whereinthe plant is a monocot plant.
 26. The fertile transgenic plant of claim25 wherein the monocot plant is a maize, barley, wheat, sorghum, rye orrice plant.
 27. The fertile transgenic plant of claim 26 wherein theplant is a maize plant.
 28. The method of claim 21 wherein the plant isa dicot plant.
 29. The method of claim 28 wherein the plant is asoybean, cotton, canola or potato plant.
 30. The fertile transgenicplant of claim 22 wherein at least a portion of the transgene insertionis altered in that it has been deleted, amplified, or re arranged. 31.The fertile transgenic plant of claim 22 wherein at least a portion ofthe transgene insertion is altered in that it has been deleted.
 32. Thefertile transgenic plant of claim 22 wherein at least a portion of thetransgene insertion is altered in that it has been amplified.
 33. A seedproduced by the fertile transgenic plant of claim 22
 34. A fertiletransgenic plant wherein at least a portion of a transgene insertion isaltered from a parent transgene insertion.
 35. The fertile transgenicplant of claim 34 , wherein the plant is hybrid.
 36. The fertiletransgenic plant of claim 34 , wherein the plant is inbred.
 37. Thefertile transgenic plant of claim 34 , wherein at least a portion of thealtered transgene insertion is altered in that it has been deleted,amplified, or rearranged.
 38. The fertile transgenic plant of claim 34 ,wherein at least a portion of the altered transgene insertion is alteredin that it has been deleted.
 39. A progeny plant of any generationcomprising an altered transgene insertion, wherein at least a portion ofthe transgene insertion is altered from the transgene insertion in aparental R0 plant.
 40. An altered transgene insertion DNA sequencepreparable by the method comprising: a) obtaining a first fertiletransgenic plant comprising for a transgene insertion DNA sequence,wherein the transgene DNA sequence comprises a pre-selected DNA sequenceflanked by directly repeated DNA sequences; b) obtaining a plurality ofprogeny of any generation of the first transgenic plant; and c)selecting a progeny fertile transgenic plant wherein at least a portionof the transgene insertion is altered as compared to the first fertiletransgenic plant.
 41. The altered transgene insertion sequence of claim40 wherein the first fertile transgenic plant is homozygous for thetransgene insertion DNA sequence.
 42. The altered transgene insertion ofclaim 40 wherein at least a portion of the transgene insertion isaltered in that it is deleted, amplified, or rearranged.
 43. The alteredtransgene insertion of claim 40 , wherein at least a portion of thetransgene insertion is altered in that it has been deleted.
 44. Thealtered transgene insertion of claim 40 , wherein expression of atransgene contained within the parental transgene insertion is altered.45. The altered transgene insertion of claim 40 where the alteration isidentified by DNA analysis.
 46. The altered transgene insertion of claim45 wherein the DNA analysis is by PCR.
 47. The altered transgeneinsertion of claim 40 wherein the plant is a monocot plant.
 48. Thealtered transgene insertion of claim 47 wherein the monocot plant is amaize, barley, wheat, sorghum, rye or rice plant.
 49. The alteredtransgene insertion of claim 48 wherein the plant is a maize plant. 50.The altered transgene insertion of claim 40 wherein the plant is a dicotplant.
 51. The altered transgene insertion of claim 50 wherein the plantis a soybean, cotton, canola, or potato plant.